CelltransmissionsThe Newsletter for Cell Signaling and Neuroscience Research

Vascular Endothelial Growth Factors (VEGFs),Their Receptors and Their InhibitionD. Stephen Charnock-Jones, University of Cambridge, United Kingdom

Application Note:

Senescent Cells Staining KitEfrat Barnea, Rina Altman, Pnina Yaish, and Dorit Zharhary Sigma-Aldrich Israel Ltd., Rehovot, Israel

In this Issue...

sigma-aldrich.com/cellsignaling

continued on page 3

Vol 21, No 1 • April 2005

C ellular senescence is a progression of eventswhereby cells move from an actively dividing

to a non-dividing state, yet remain metabolicallyactive. The cellular senescence process is associ-ated with aging and age-related diseases includ-ing atherosclerosis, osteoarthritis, immune senes-cence, skin aging, Alzheimer’s dementia andcancer. The decrease in cell division is virtuallyirreversible and complete. In conjunction withthe loss of the ability to divide, changes occur inthe morphology, shape, and physical appearanceof the cells, and in their pattern of gene expres-sion. Although the cells may remain viable for a

long time, at the end of the process cell deathusually occurs. The Senescent Cells Staining Kit(Prod. Code CS0030) is designed for identifyingsenescent cells using a simple and rapid stainingprocedure.

continued on page 7

V ascular endothelial growth factor A (VEGF-A)is a member of a family of potent angio-

genic growth factors which together play criticalroles in determining the structure and functionof blood vessels. Individual members of thisfamily are essential for the differentiation ofendothelial cells, the formation of primitiveendothelial cells into capillary like tubes, themaintenance of mature capillaries, the differenti-ation, generation and maintenance of thelymphatic endothelium, and the response ofendothelial cells to proliferative stimuli. As such,these growth factors play an essential role inblood vessel development. In addition, they areimplicated in physiological angiogenesis and inparticular, in numerous pathologies in whichangiogenesis is a key feature.

There are five members of this gene family inmammals. They all share certain commonfeatures and will be described in a later sectionof this review. The proteins act as homodimericstructures with molecular weights rangingbetween approximately 35 kDa and 80 kDa.The homology between the family members ishighest in the central region (referred to as theVEGF homology domain) and this region formsa cysteine knot structure. This type of structureis also found in platelet derived growth factor(PDGF).

The archetypal and best characterized memberof the VEGF family is VEGF-A, the sequence ofwhich was first described in 1989 [1]. Thesequence of another factor, named vasculature

New Products pp. 12-15

Assay Kits• Phosphospecific ELISAs• FAM-FLICA/Caspase Assays• Tyrosine Kinases• Plus many more! p. 11

Antibodies to• Sonic Hedgehog p. 9

• AKR1C3 p. 16

• β-Amyloid [13-28] p. 16

• Apolipoprotein J p. 17

• Frizzled Antibodies p. 18

• Potassium Channel KCNK9 (TASK 3) p. 18

• Importin α1 p. 22

• PINCH-1 p. 22

• Plus many more! p. 12

Available First from Sigma-RBI!• Ondansetron and

UCM-17197: 5-HT3 serotoninreceptor antagonists p. 17

• NNC 55-0396: T-type Ca2+

channel blocker p. 19

• Psora-4: Kv1.3 K+-channelblocker p. 19

Panorama Human ProteinFunction Microarray - p53 p. 8

Jervine: Sonic hedgehoginhibitor p. 9

TOFA: Acetyl-CoA carboxylase inhibitor p. 9

Paroxetine maleate: Serotoninreuptake inhibitor p. 17

PD 81,723: Allosteric enhancerof A1 adenosine receptors p. 18

PNU-37883A: ATP-sensitive K+

Kir6.1/SUR2B channel blocker p. 19

A-350619: Novel, solubleguanylyl cyclase activator p. 20

• Contains all the reagents required foridentifying senescent cells

• Simple and rapid staining procedure basedon histochemical stain for β-galactosidase

• Sufficient for at least 100 tests in 35-mmcell culture plates

Need a better way to assay?

Think Insidethe Box.

Get your next Cell Signaling Assay Kit FREE.

When is it a good time for researchers to think

inside the box? When it comes to choosing the

best tools for making your daily research easier. After all,

you have better ways to spend your time than developing

and validating cell signaling assays. Let our customized

kits do the work for you. With more than 100 available,

we’re sure to have what you need right now.

Order your kit at: sigma-aldrich.com/assaykits_oj6

After you use the kit, all we ask is that you share your

results with us. When you do, you’ll automatically

qualify for a second kit, absolutely free. We want to

hear about your results, because those insights will be

invaluable as we continue to create new ways to make

science easier for you.

Our assay kits are just one of the many cell signaling

solutions we offer to more than a million scientists,

every day. From the widest selection of products to the

best technical support, we’re the research partner of

choice around the world. When you need a better way

to assay, we’re the only partner you need.

Qualify for your FREE kit today at: sigma-aldrich.com/assaykits_oj6

sigma-aldrich.com

®

®L E A D E R S H I P I N L I F E S C I E N C E , H I G H T E C H N O L O G Y A N D S E R V I C ESIGMA-ALDRICH CORPORATION • BOX 14508 • ST. LOUIS • MISSOURI 63178 • USA

Purchase Any One ofThese Kits, and YourNext Kit is FREE!• Total and Phosphospecific

ERK ELISAs

• Total and Phosphospecific Bcl-2 ELISAs

• Total and Phosphospecific Tau ELISAs

• Senescent Cell Staining Kit

• BACE1 Activity Assay Kit

• JNK/p38/MEK Activity Assay Kit

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

3

VEG

Fs, T

hei

r R

ecep

tors

an

dTh

eir

Inh

ibit

ion

permeability factor (VPF), was described in the samejournal and has the same sequence as VEGF [2]. These twopapers coincidentally revealed two of the principle ways inwhich VEGF-A acts as an endothelial growth factor and asa vasculature permeability-promoting factor. These andsubsequent papers revealed that the VEGF-A gene canproduce several alternatively spliced transcripts, each ofwhich has slightly different properties, and that this subtlevariation in structure has functional consequences for theway blood vessel growth is regulated [3]. The mostcommon forms of VEGF-A, named on the basis of thenumber of amino acids in each of the two chains formingthe homodimeric structure, are VEGF-A121, VEGF-A145,VEGF-A165, VEGF-A189 and VEGF-A206 (see Figure 1).More recent papers indicate the existence of other splicevariants and, indeed, proteolytically cleaved products thatstill retain some biological activity have been identified [4].

VEGF-A is undoubtedly essential for vasculogenesis andangiogenesis in the embryo. This is clearly revealed bymouse knockout studies in which deletion of a single alleleof the VEGF-A gene is sufficient to cause embryonic death[5,6]. The fact that heterozygous deletion is lethal suggeststhat the endothelial cells, which respond to VEGF, areexquisitely sensitive to the local level of VEGF-A and thateven a modest reduction of this is sufficient to induce pro-found biological effects. Since its original description, VEGFhas been shown to have effects on a variety of non-endothelial cells, including alveolar epithelial cells andhematopoietic stem cells [7]. Both of these cell typesrespond to VEGF-A and, in the case of the hematopoieticcells, are therapeutic targets [8].

Physiological angiogenesis in the adult is generally a rareevent. However, in the normal ovarian cycle of the femalethere is profound angiogenesis as the corpus luteumdevelops. Blocking VEGF-A action in this tissue abolishesangiogenesis and leads to the failure of luteal growth [9].

These and other studies indicate the pivotal role of VEGF-Ain physiological angiogenesis. Aberrant angiogenesis is acharacteristic of several clinically important conditions andas a result there has been considerable interest in under-standing the factors that regulate pathological angio-genesis. The two conditions for which the most informa-tion has been gained relate to the angiogenesis thatoccurs in solid tumors and the aberrant vessel growthcharacteristic of several retinopathies. In both instances,there is compelling evidence that VEGF-A plays a centralrole and inhibition of the actions of VEGF-A can reduceaberrant growth.

Numerous studies have been reported that describe theincrease in VEGF-A protein and mRNA in tumors. Indeedmany of these studies show a correlation between themicrovascular density in the tumor and the level of VEGF-like immunoreactivity [10-13]. The first study describingthe functional consequences of blocking the actions ofVEGF-A were published in 1993 [14]. The authors demon-strated that the growth of a variety of tumor cell lines,which was unaffected in vitro by anti-VEGF-A antibodytreatment, was inhibited by between 70 and 90% in vivo.This antibody (A.4.6.1) has subsequently been humanizedand developed for clinical use [15,16]. Another elegantstudy demonstrated that flk/KDR (one of the VEGF recep-tors) was pivotal for the VEGF response. Retroviral-mediat-ed transduction of tumor-bearing mice with a truncated(i.e. a dominant-negative) receptor profoundly inhibitedtumor growth. The identification of the importance of thisreceptor has subsequently led to the development of smallmolecules to selectively target this receptor. VEGF-A is alsoa survival factor for endothelial cells and this has beenelegantly shown in vivo [17]. VEGF-A driven angiogenesisin the eye has been reversed in animal models and this hasrecently been extended to human patients [18,19].

VEGF Family MembersAs already mentioned, VEGF-A is the prototypical memberof a family of related growth factors. Those so fardescribed in humans, with limited, but concordant data inother species, are placenta growth factor (PlGF), VEGF-B,VEGF-C and VEGF-D [20-23]. An additional molecule,VEGF-E has been described, but this is in fact encoded byan orf virus which infects sheep. Such infections result inhighly vascularized lesions on the face and mouth of thesheep and this virally encoded protein is able to stimulatehost and endothelial cell proliferation, hence, the highlyvascularized lesions. However, while this protein has bothstructural and functional similarity to the mammalianVEGFs, it is not a mammalian protein [24].

The function and biological importance of these relatedgrowth factors are less well defined than for VEGF-A, butthere are clear data indicating that PlGF is able to inducethe mobilization of bone marrow-derived endothelial pre-cursors in a similar way to VEGF-A and that, while it is notrequired for embryonic growth and development, PlGF

Vascular Endothelial Growth Factors, Their Receptors and Their InhibitionD. Stephen Charnock-Jones (continued from cover)

VEGF-A206

VEGF-A189

VEGF-A183

VEGF-A165

VEGF-A145

VEGF-A121

VEGF-A110

EXON 1-5 6 6' 7 8

Figure 1. VEGF Ligands: Gene structure of the VEGF-A gene and the principalsplice variants giving rise to VEGF-A121, VEGF-A145, VEGF-A165, VEGF-A189 andVEGF-A206. An additional form containing 110 amino acids is generated byproteolytic cleavage.

plays an important role in pathological angiogenesis. Forexample, in PlGF null mice, tumor angiogenesis andcollateral formation following ligation of the femoral arteryare both severely compromised [25]. VEGF-B is not essen-tial for embryonic development, but plays a role in cardiacdevelopment and VEGF-B null mice demonstrate animpaired response to experimentally induced myocardialischemia [26].

VEGF-C has been shown to play an essential role in theformation of the lymphatic endothelium, and deletion ofthis gene in mice causes a lethal phenotype with theembryos exhibiting profound edema and a marked lack oflymphatics [27]. VEGF-D is implicated in lymphangiogenesisand is up-regulated in a variety of tumors [28,29]. Alter-natively spliced transcripts encoding different forms of theseVEGF related genes also exist and these are illustrated inFigure 1. In addition to alternative splicing, VEGF-C inparticular is extensively proteolytically processed prior toformation of the active molecule [30].

VEGF ReceptorsThree high affinity tyrosine kinase receptors exist for theVEGFs and are referred to as VEGF-R1 (fms-like tyrosine-kinase, flt-1), VEGF-R2 (kinase domain receptor, KDR inhumans or fetal liver kinase, flk in mouse) and VEGF-R3(flt-4). Neuropilin 1, which plays a role in neuronal guid-ance and binds members of the semaphorin family, bindssome of the splice variants of VEGF-A and, in a mannerthat is currently poorly understood, facilitates VEGF-A165signaling through VEGF-R2 [31]. The tyrosine kinasereceptors were all initially cloned as orphan receptors andshare a broadly similar structure. VEGF-R1, VEGF-R2 andVEGF-R3 all contain seven immunoglobulin-like repeats intheir extracellular domains, a single membrane spanningdomain and a split tyrosine kinase domain within the intra-cellular portion of the protein [32-34].

Soluble variants of VEGF-R1 (sVEGF-R1) that have beenreported are generated by alternative splicing. These retainvarying amounts of the extracellular ligand binding domain,but lack the transmembrane and tyrosine kinase domains[35,36]. They still bind VEGF-A, PlGF and VEGF-B, but areunable to signal. Thus, sVEGF-R1 is a competitive antagonistand in some circumstances may act as a dominant negativeby forming heterodimers on the cell surface with kinase-active receptors. sVEGF-R1 is produced at low levels byendothelial cells, but is highly expressed in the placenta [37].

While these receptors share similar overall architecture,they do show a considerable degree of ligand selectivity assummarized in Figure 2. VEGF-A, VEGF-B and PlGF all bindwith high affinity to VEGF-R1; VEGF-A, VEGF-C and VEGF-D bind with high affinity to VEGF-R2; VEGF-C and VEGF-Dbind with high affinity to VEGF-R3 and VEGF-E binds toonly VEGF-R2.

The importance of each of these receptors to vessel growthand maintenance has been clearly demonstrated in knock-out and other manipulative models. For example, inactiva-tion of the VEGF-R2 gene in mice prevents the formationof endothelial cells. This indicates that activation of thisreceptor is an essential step in the differentiation ofendothelial precursors (so called haemangioblast) [38].Inactivation of VEGF-R1 is lethal at a similar time of devel-opment (approximately day 9.5 of gestation). However, inthis instance, endothelial cells form and proliferate exten-sively, but are unable to form well organized functionalvessels [39]. Deletion of VEGF-R3 also produces an embry-onic lethal phenotype with the embryos dying at approxi-mately embryonic day 9.5 due to fluid accumulation in thepericardial cavity and subsequent cardiovascular failure[40]. These and other animal studies confirms the impor-tance of the VEGF family and, in particular, the role VEGF-A plays in blood vessel growth, development andmaintenance. VEGF-A in particular has also been shown toplay a pivotal role in numerous animal models of significantpathologies and there is good correlative data with thecorresponding human conditions.

The idea that blood vessel growth is required for solidtumor growth is not a new concept and this literature hasrecently been reviewed [41]. An insight that has proveninstrumental in the development of this field was made in1971. In his seminal paper, Judah Folkman suggested thatblocking tumor angiogenesis may be an effective way toprevent tumor growth and metastasis [42]. This hypothesishas been verified through numerous mouse studies inwhich the actions or production of VEGF-A have beeninhibited [14,41,43]. There has therefore been a greatcommercial drive to develop VEGF receptor antagonists forclinical use in the treatment of certain cancers in humans.

One approach is to inhibit VEGF production using anti-sense or siRNA coupled with some form of gene therapydelivery system. These are extremely complex approachesand only modest progress has been made to date in thisarea. Researchers have taken a more productive approachby inhibiting the action of VEGF-A. The most obvious ofthese has used a humanized monoclonal antibody whichbinds VEGF-A and prevents its interaction with cell surfacereceptors. This approach was adopted by Genentech whodemonstrated in 1993 that monoclonal antibody treatmentof tumor-bearing mice resulted in a significant inhibition oftumor growth [14]. Since that time, this approach has beendeveloped further and the resultant drug, bevacizumab,Avastin®, has successfully completed phase III clinical trialsand shows considerable potential for human therapy [15].Other strategies are also being explored to bind and inacti-vate VEGF, for example, using truncated forms of highaffinity receptors. These are variants of natural solubleforms of the receptors or recombinant chimeric moleculessuch as the “VEGF trap” [44]. This approach has beenadopted by Regeneron and their clinical development can-didate is currently in phase I/II trials. A more unexpected,but seemingly successful approach has involved the use of

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

4V

EGFs

, Th

eir

Rec

epto

rs a

nd

Thei

r In

hib

itio

nVascular Endothelial Growth Factors... (continued)

an RNA adaptomer which specifically binds VEGF and pre-vents it from interacting with its cell surface receptor. Thisapproach has been used by Eyetech Pharmaceuticals andthe drug candidate, Macugen®, is currently in phase II/IIIclinical trials [19,45].

Several different approaches focus on the receptor ratherthan the ligand; the most obvious of which is to use anantibody which binds to the receptor, thereby preventingligand binding or receptor dimerization. This approach hasbeen developed by Imclone (for example, targeting VEGF-R2,with IMC-1121b, currently in phase I) and there are alsogene therapy type of approaches using ribozymes thattarget one or other of the VEGF receptors (Angiozyme®,Ribozyme Pharmaceuticals Inc). The most popular approach,and probably the one that is the most serious competitionto the humanized monoclonal antibody strategy, is todevelop small molecules that specifically inhibit the tyrosinekinase action of one or more of the VEGF receptors. This isan attractive strategy since the receptors are specific for theVEGFs, and upon ligand binding become active kinases. Ifthis enzymatic activity could be inhibited, then the biologicaleffects of VEGF would be abolished. Such drugs would notnecessitate the complex manufacturing or delivery systemsrequired by so-called “biologicals” or DNA/RNA basedapproaches. However, the VEGF receptors, in addition tobeing closely related to each other, are structurally similar toseveral other tyrosine kinases, in particular, the PDGF recep-tor and c-kit. This makes the development of truly selectiveinhibitors a significant challenge. Nonetheless, a large num-ber of companies have developed compounds which are

useful for biomedical research and some of them are beingconsidered for clinical use. These are summarized in Table 1.The most widely used VEGF receptor inhibitor of this type isSU5416 (developed originally by Sugen, but now owned byPfizer). This was the first member of this class of compoundsto be developed, although unfortunately it was shown topossess several undesirable characteristics. More recentlydeveloped inhibitors demonstrate greater selectivity withimproved pharmacological characteristics and may thereforeprove to be more effective in the clinic [47,48].

Agent Specificity Company Phase

PTK787 VEGF-R1 and R2 Novartis IIIZD 6474 VEGF-R1 and R2 AstraZeneca IIAG 013676 Several RTKs Pfizer IISU11248 Several RTKs Pfizer I/II

In conclusion, the VEGF family of growth factors plays anessential role in both physiological and pathological angio-genesis. Numerous inhibitors have been developed tointerfere with the biological actions of members of thisfamily. This has led to both the elucidation of the mode ofaction of the VEGFs and the development of potentiallynovel therapies for the treatment of cancer and eye disease.

References1. Leung, D.W., et al., Science, 246, 1306-1309 (1989).2. Keck, P.J., et al., Science, 246, 1309-1312 (1989).3. Houck, K.A., et al., Mol. Endocrinol., 5, 1806-1814 (1991).4. Keyt, B.A., et al., J. Biol. Chem., 271, 7788-7795 (1996).5. Ferrara, N., et al., Nature, 380, 439-442 (1996).6. Carmeliet, P., et al., Nature, 380, 435-439 (1996).7. Compernolle, V., et al., Nat. Med., 8, 702-710 (2002).8. Rafii, S., et al., Nat. Rev. Cancer, 2, 826-835 (2002).9. Ferrara, N., et al., Nat. Med., 4, 336-340 (1998).10. Weidner, N., et al., New Engl. J. Med., 324, 1-8 (1991).11. Obermair, A., et al., J. Natl. Cancer Inst., 89, 1212-1217 (1997).12. Volm, M., et al., Anticancer Res., 16, 213-217 (1996).13. Chaudhary, R., et al., Anticancer Res., 19, 3479-3484 (1999).14. Kim, K.J., et al., Nature, 362, 841-844 (1993).15. Hurwitz, H., et al., New Engl. J. Med., 350, 2335-2342 (2004).16. Ferrara, N., et al., Nat. Rev. Drug Discov., 3, 391-400 (2004).17. Alon, T., et al., Nat. Med., 1, 1024-1028 (1995).18. Aiello, L.P., et al., Proc. Natl. Acad. Sci. USA, 92, 10457-10461 (1995).19. EyetechStudyGroup, Ophthalmology, 110, 979-986 (2003).20. Maglione, D., et al., Proc. Natl. Acad. Sci. USA, 88, 9267-9271 (1991).21. Olofsson, B., et al., Proc. Natl. Acad. Sci. USA, 93, 2576-2581 (1996).22. Joukov, V., et al. [published erratum appears in EMBO J., 15, 1751 (1996)],

EMBO J., 15, 290-298 (1996).23. Orlandini, M., et al., Proc. Natl. Acad. Sci. USA, 93, 11675-11680 (1996).24. Ogawa, S., et al., J. Biol. Chem., 273, 31273-31282 (1998).25. Carmeliet, P., et al., Nat. Med., 7, 575-583 (2001).26. Bellomo, D., et al., Circ. Res., 86, E29-E35 (2000).27. Karkkainen, M.J., et al., Nat. Immunol., 5, 74-80 (2004).28. Stacker, S.A., et al., Nat. Med., 7, 186-191 (2001).29. White, J.D., et al., Cancer Res., 62, 1669-1675 (2002).30. Joukov, V., et al., EMBO J., 16, 3898-3911 (1997).31. Soker, S., et al., Cell, 92, 735-745 (1998).32. Shibuya, M., et al., Oncogene, 5, 519-524 (1990).33. Terman, B.I., et al., Biochem. Biophys. Res. Commun., 187, 1579-1586 (1992).34. Pajusola, K., et al., Cancer Res., 52, 5738-5743 (1992).35. Kendall, R.L., et al., Proc. Natl. Acad. Sci. USA, 90, 10705-10709 (1993).36. Boocock, C.A., et al., J. Natl. Cancer Inst., 87, 506-516 (1995).37. Clark, D.E., et al., Biol. Reprod., 59, 1540-1548 (1998).38. Shalaby, F., et al., Nature, 376, 62-66 (1995).39. Fong, G.H., et al., Nature, 376, 66-70 (1995).40. Dumont, D.J., et al., Science, 282, 946-949 (1998).41. Ferrara, N., Nat. Rev. Cancer, 2, 795-803 (2002).42. Folkman, J., New Engl. J. Med., 285, 1182-1186 (1971).43. Millauer, B., et al., Nature, 367, 576-579 (1994).

Vascular Endothelial Growth Factors... (continued)

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

5

VEG

Fs, T

hei

r R

ecep

tors

an

dTh

eir

Inh

ibit

ion

Table 1. VEGF Receptor Tyrosine Kinase inhibitors in clinical trials for treatmentof cancer.

ss

sVEGF-R1

VEGF-R1 VEGF-R2

VEGF-R3

VEGF-APIGFVEGF-B

VEGF-AVEGF-CVEGF-DVEGF-E

VEGF-CVEGF-D

Figure 2. VEGF Receptors. The VEGF receptor family consists of three trans-membrane tyrosine kinase receptors: VEGF-R1 (flt-1), VEGF-R2 (KDR in manflk-1 in mouse) and VEGF-R3 (flt-4). Several naturally occurring soluble formsof VEGF-R1 have been described. VEGF-R1 and VEGF-R2 have seven extra-cellular immunoglobulin-like domains, but in VEGF-R3 the fifth Ig-like domainis cleaved into disulfide-linked subunits. The specificity of ligand binding isindicated by arrows above the receptors.

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

6V

EGFs

, Th

eir

Rec

epto

rs a

nd

Thei

r In

hib

itio

n

References (continued)

44. Huang, J., et al., Proc. Natl. Acad. Sci. USA, 100, 7785-7790 (2003).45. Nimjee, S.M., et al., Annu. Rev. Med., 56, 555-583 (2005).46. Sun, L., et al., J. Med. Chem., 43, 2655-2663 (2000).47. Underiner, T.L., et al., Curr. Med. Chem., 11, 731-745 (2004).48. Boyer, S.J., Curr. Top. Med. Chem., 2, 973-1000 (2002).

About the AuthorSteve Charnock-Jones received his Ph.D. from the University ofCambridge in the UK. He then carried out his postdoctoral researchin the laboratory of Dr. Sydney Brenner within the Medical ResearchCouncil's Molecular Genetics Unit in Cambridge. Dr. Charnock-Jones is currently University Reader in the Department of Obstetricsand Gynecology at the University of Cambridge where his researchfocuses on reproductive angiogenesis.

Vascular Endothelial Growth Factors.. (continued)

Antibodies

V 1253 Anti-Vascular Endothelial Growth Factor A (goat)

V 6627 Anti-Vascular Endothelial Growth Factor A (goat)

V 1010 Anti-Vascular Endothelial Growth Factor B 167/186(goat)

V 1264 Anti-Vascular Endothelial Growth Factor C (goat)

V 6134 Anti-Vascular Endothelial Growth Factor D (goat)

V 0885 Anti-Vascular Endothelial Growth Factor D (goat)

V 1139 Anti-Vascular Endothelial Growth Factor Receptor-1(goat)

V 1014 Anti-Vascular Endothelial Growth Factor Receptor-2(goat)

V 2884 Anti-Vascular Endothelial Growth Factor Receptor-3(goat)

V 1135 Anti-Vascular Endothelial Growth Factor Receptor-3(goat)

V 4758 Monoclonal Anti-Vascular Endothelial Growth Factor(mouse), Clone 26503.11

V 4762 Monoclonal Anti-Vascular Endothelial Growth FactorReceptor-1 (mouse), Clone FLT-19

V 4262 Monoclonal Anti-Vascular Endothelial Growth FactorReceptor-1 (mouse), Clone FLT-11

V 9134 Monoclonal Anti-Vascular Endothelial Growth FactorReceptor-2 (mouse), Clone KDR-1

P 3868 Monoclonal Anti-Placenta Growth Factor (mouse),Clone 37203.111

P 3493 Monoclonal Anti-Placenta Growth Factor-2 (mouse),Clone 62526.111

V 5014 Anti-phospho-VEGF Receptor-2 [pTyr1054/pTyr1059](rabbit)

V 5389 Anti-phospho-VEGF Receptor-2 [pTyr1054] (rabbit)

V 5264 Anti-phospho-VEGF Receptor-2 [pTyr1214] (rabbit)

V 5239 Anti-phospho-VEGF Receptor-2 [pTyr951] (rabbit)

Receptors

V 1385 VEGF Receptor-1 (Flt-1)/Fc Chimera, human

V 6137 VEGF Receptor-1 (Flt-1)/Fc Chimera, mouse

V 6758 VEGF Receptor-2 (Flk-1, KDR)/Fc Chimera, human

V 6883 VEGF Receptor-2 (Flk-1, KDR)/Fc Chimera, mouse

V 1260 VEGF Receptor-3 (Flt-4)/Fc Chimera, human

V 6633 VEGF Receptor-3 (Flt-4)/Fc Chimera, mouse

Proteins

P 1588 Placenta Growth Factor, human, recombinant,expressed in E. coli

P 5739 Placenta Growth Factor-2, mouse, recombinant,expressed in Sf 21 cells

V 7259 Vascular Endothelial Growth Factor, human,recombinant, expressed in E. coli

V 4512 Vascular Endothelial Growth Factor, mouse,recombinant, expressed in E. coli

V 6012 Vascular Endothelial Growth Factor D human,recombinant, expressed in Sf 21 cells

Inhibitors

L 2400 Lavendustin AInhibits VEGF-induced angiogenesis.

T 4192 SU1498Potent and selective VEGF receptor kinase, Flk-1 inhibitor;very weak PDGFR-kinase, EGFR-kinase and HER-2 kinaseinhibitor.

S 8567 SU4312Vascular endothelial growth factor (VEGF) receptor proteintyrosine kinase 1/2 and platelet derived growth factor(PDGF) receptor inhibitor.

S 8442 SU5416Vascular endothelial growth factor (VEGF) receptor proteintyrosine kinase 1/2 inhibitor.

T 0318 TranilastInhibits VEGF-induced angiogenesis in vivo and has alsobeen shown to inhibit proliferation and tube formation ofhuman endothelial cells in vitro.

T 5317 Tyrphostin AG 1433PDGFβ receptor tyrosine kinase inhibitor; inhibits VEGF-induced angiogenesis.

Vascular Endothelial Growth Factor (VEGF) Products Available from Sigma-RBI

Visit Sigma-Aldrich at:

Experimental BiologySan Diego, California

April 2-6, 2005Booth Nos. 1216 and 1218

American Association for Cancer Research (AACR)

Anaheim, CaliforniaApril 16-20, 2005Booth No. 1404

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

7

Sen

esce

nt

Cel

ls S

tain

ing

Kit

Principle of the AssayThe kit is based on a histochemical stain for β-galacto-sidase activity, at pH 6, which is unique to senescent cells.The activity at pH 6 is easily detectable in senescent cells,but undetectable in quiescent, immortal or tumor cells [1].As a control, cells are also stained at acidic pH 4 whereeukaryotic lysosomal β-galactosidase is active in all cells.

ProcedureSeveral types of cells representing normal and senescentcells were used as a model for illustrating the principle ofthe kit.Normal Cells• Human Foreskin Fibroblasts (HFF) primary cells at an

early passage (passage 5).• pIND-p53 cells - H 1299 cells stably transfected with

Muristerone A induced p53 gene [2].Senescent Cells• Human Foreskin Fibroblasts (HFF) primary cells at a late

passage (passage 28).• Muristerone A induced pIND-p53 cells - H 1299 cells sta-

bly transfected with Muristerone A induced p53 gene[2]. Induction of p53 expression induces senescence inthe cells within 4-5 days.

Control Cells • HeLa cells, transfected or non-transfected with a

bacterial β-galactosidase reporter plasmid.• Muristerone A treated HeLa cells.

Cells were grown in 35-mmtissue culture plates or a 96-wellplate and treated according tothe procedure illustrated inFigure 1 using the SenescentCells Staining Kit (Prod. CodeCS0030). The cells were fixedand incubated at 37°C in a pH 6staining buffer, containing anartificial β-galactosidase substrate(X-gal). As a control experiment,the fixed cells were stained withstaining buffer at pH 4 where thelysosomal β-galactosidase isactive, or at pH 7.5, where onlythe bacterial β-galactosidase intransfected cells is active.

ResultsTable 1 summarizes the staining data of the senescentand control cells at pH 4, pH 6 and pH 7.5. As expected,at pH 4 all the cells tested stained blue due to the activityof the mammalian lysosomal β-galactosidase that is activeat pH 4. At pH 7.5 only HeLa cells expressing the bacterial β-galactosidase stained blue because this is the optimalpH for the bacterial β-galactosidase. Blue staining at pH 6was observed only in HFF cells at a late passage (Figure2B) or in Muristerone A induced pIND-p53 cells. Therewas no staining in HFF at an early passage (Figure 2A), innon-induced pIND-p53 cells or in HeLa cells. These resultsindicate that β-galactosidase staining at pH 6 occurs onlyin senescent cells.

DiscussionCleavage of X-gal by β-galactosidase at pH 6.0 leading toa blue staining of cells was found to be specific for senes-cent cells, while this cleaving activity is not detectable atpH 6.0 in pre-senescent cells whether they are growing orimmortal (cell line). Staining of cells using the SenescentCells Staining Kit (Prod. Code CS0030), as describedabove, allows fast and easy detection of senescent cellsand provides a tool for distinguishing between senescentand pre-senescent cells.

References1. Dimri, G.P., et al., Biomarker that identifies senescent human cells in culture

and in aging skin in vivo. Proc. Natl. Acad. Sci. USA, 92, 9363-9367 (1995).2. Wang, Y., et al., Induced p53 expression in lung cancer cell line promotes

cell senescence and differentially modifies the cytotoxicity of anti-cancerdrugs. Oncogene, 17, 923-930 (1998).

Manufactured under license to US Patent Nos. 5,491,069 and 5,795,728.

Ordering InformationProduct Description Unit

CS0030 Senescent Cells Histochemical Staining Kit 1 kit

Cells

Cell wash

Cell fixation

Cell wash

Stained cells

MicroscopyVisualization

Incubation withStaining Buffer

Non-induced Induced Muristerone A HeLa Cellular HFF HFF pIND-p53 pIND-p53 treated β-gal

pH Passage 5 Passage 28 H 1299 H 1299 HeLa HeLa transfected4 + + + + + + +6 – + – + – – –

7.5 – – – – – – +

Figure 1. Flow chart of thestaining procedure.

Table 1. β-galactosidase activity in various cell types

A) B)

Figure 2. Detection of senescent cells by β-galactosidase staining. HumanForeskin Fibroblasts (HFF) primary cells at an early passage. (A: 5 passages) and at a latepassage (B: 28 passages) were stained using the Senescent Cell Staining Kit (Prod. CodeCS0030). The HFF cells at passage 28 show a blue staining indicating that these aresenescent.

Senescent Cells Staining Kit continued from page 1

The appearance of a blue color in cells, due to β-galactosidase activity, is indicated by (+). The cells were incubated with an X-gal substrate containing staining buffer at different pHs (4, 6and 7.5). β-galactosidase staining representing activity specific for senescent cells is highlighted in red.

• Suitable for DNA, proteins, and small molecule studies• 49 human p53 variants• Uniformly oriented & correctly folded proteins

sigma-aldrich.com L E A D E R S H I P I N L I F E S C I E N C E , H I G H T E C H N O L O G Y A N D S E R V I C ESIGMA-ALDRICH CORPORATION • BOX 14508 • ST. LOUIS • MISSOURI 63178 • USA

A significant technical challenge associated with protein arrays isensuring the full functionality and biological availability of

spotted proteins. Without technologies that confirm biological activity,profiles cannot accurately reflect protein interaction or function.Sigma’s novel protein array platform technology eliminates the doubtby ensuring only correctly folded and fully functional proteins areimmobilized. Confidently identify relevant p53 interactions withPanorama Human Protein Function Microarray – p53.

• Proprietary BCCP (biotin-carboxyl carrier protein) tagging technologyensures only fully functional p53 proteins are immobilized

• Accurate representation of 49 p53 varients. Configured in two sub-arrays per slide with each protein spotted in duplicate

• Compatible with probing by DNA, proteins, kinases and smallmolecules and with Cy3/Cy5-based detection chemistries

Product Code Description

HPFM1 Panorama Human Protein Function Microarray - p53

Learn more about Panorama Human Protein Function Microarray - p53 at:sigma-aldrich.com/p53ct

p53 PROTEIN INTERACTIONS THAT COUNT!The First Fully Functional p53 Protein Array

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

9

New

Pro

du

ct H

igh

ligh

ts

Jervine: Sonic hedgehog (Shh) signaling pathwayinhibitor; cyclopamine analog

Cell-permeable steroidal alkaloidwith teratogenic effects; inducescyclopia by blocking Shh signaling(IC50 ~500–700 nM in s12 cells).

References1 Williams, J.A., et al., Identification of a small molecule inhibitor of the

hedgehog signaling pathway: effects on basal cell carcinoma-like lesions.Proc. Natl. Acad. Sci. USA, 100, 4616-4621 (2003).

2. Mistretta, C.M., et al., Cyclopamine and jervine in embryonic rat tonguecultures demonstrate a role for Shh signaling in taste papilla developmentand patterning: fungiform papillae double in number and form in novellocations in dorsal lingual epithelium. Dev. Biol., 254, 1-18 (2003).

Prod. Code J 4145

HO

H3C

O

H3C

NHCH3

H3C

O

Prod. Code: S 4944Clone Name: 171018Product Form: Purified rat immunoglobulinImmunogen: Purified, E. coli-derived, recombinant mouse Sonic

hedgehog (Shh) N-terminal peptide (amino acids25-198)

Isotype: rat IgG2aSpecies Cross Reactivity: Human and mouse Shh

Sonic hedgehog (Shh) is an important cell signaling moleculeexpressed during embryonic development. Shh is involved in thepatterning of the developing embryonic nervous system, somiteand limb. The N-terminal peptide of Shh is released by auto-proteolysis and functions through interactions with a multi-component receptor complex containing the transmembraneproteins, Patched and Smoothened. Shh protein is expressed inkey embryonic tissues such as the Hensen’s node, zone of polar-izing activity in the posterior limb bud, notochord, and floorplate of the neural tube.

Applications: Immunoblotting, immunohistochemistry,neutralization, and ELISA (capture)

References1. Echelard, Y., et al., Sonic hedgehog, a member of a family of putative sig-

naling molecules, is implicated in the regulation of CNS polarity. Cell, 75,1417-1430 (1993).

2. Weed, M., et al., The role of sonic hedgehog in vertebrate development.Matrix Biol., 16, 53-58 (1997).

3. Ingham, P.W. and McMahon, A.P., et al., Hedgehog signaling in animaldevelopment: paradigms and principles. Genes Dev., 15, 3059-3087 (2001).

Monoclonal Anti-Sonic Hedgehog(Shh), N-Terminal

Hedgehog-interacting Protein (Hip), mouse,recombinant, expressed in NSO cells

Prod. Code H 5163

Transcriptional target in hedgehog signaling; novel regulatoryprotein that binds with all three mammalian hedgehogs (Shh,Dhh, and Ihh). The biological activity is measured by its ability toinhibit Sonic hedgehog (Shh) induction of alkaline phosphataseproduction in C3H10T1/2 fibroblasts.

References1. Chuang, P.T. and McMahon, A.P., Vertebrate hedgehog signaling modulat-

ed by induction of a hedgehog-binding protein. Nature, 397, 617-621 (1999).2. Ingham, P.W. and McMahon, A.P., et al., Hedgehog signaling in animal

development: paradigms and principles. Genes Dev., 15, 3059-3087 (2001).

Related Products

Product Name Description Prod. Code

Cyclopamine Hedgehog signaling pathway inhibitor C 4116

Sonic Hedgehog Peptide, mouse, recombinant S 0191

TOFA: Potent, reversible, cell-permeable andcompetitive acetyl-CoA carboxylase (ACC) inhibitor

Prod. Code T 6575

Acetyl-CoA carboxylase (ACC) is a keyenzyme involved in fatty acid bio-synthesis. TOFA has been shown to

dose-dependently inhibit fatty acid synthesis in human breastcancer cell line MCF7 displaying an IC50 value of 4 µM.

References1. Landree, L.E., et al., C75, a fatty acid synthase inhibitor, modulates AMP-

activated protein kinase to alter neuronal energy metabolism. J. Biol.Chem., 279, 3817-3827 (2004).

2. Thupari, J.N., et al., Fatty acid synthase inhibition in human breast cancercells leads to malonyl-CoA-induced inhibition of fatty acid oxidation andcytotoxicity. Biochem. Biophys. Res. Commun., 285, 217-223 (2001).

OHOOC O(CH2)13CH3

Sodium stibogluconate: Irreversible proteintyrosine phosphatase (PTP) inhibitor

Prod. Code S 5319

Inhibits in vitro PTPases Src homolo-gy PTPase1 (SHP-1; 10 µg/ml), SHP-2 (100 µg/ml), and PTP1B (100µg/ml), but not mitogen-activatedprotein kinase phosphatase-1(MKP1), a dual-specificity phos-

phatase. Inhibition of activity was determined by measuringdephosphorylation of a synthetic phosphotyrosine peptide by theGST/phosphatase fusion protein [1]. Routinely used for the treat-ment of Leishmaniasis in humans, a parasitic disease spread bythe bite of sand flies infected with the protozoa Leishmaniadonovani [2].

References1. Pathak, M.K. and Yi, T., Sodium stibogluconate is a potent inhibitor of

protein tyrosine phosphatases and augments cytokine responses in hemo-poietic cell lines. J. Immunol., 167, 3391-3397 (2001).

2. Wyllie, S., et al., Dual action of antimonial drugs on thiol redox metabo-lism in the human pathogen Leishmania donovani. J. Biol. Chem., 279,39925-39932 (2004).

Related Products

Product Name Descriptor Prod. Code

Dephostatin CD45-associated tyrosine D 8065phosphatase inhibitor

NCS 95397 Cdc2 phosphatase inhibitor N 1786

Sodium orthovanadate Tyrosine phosphatase inhibitor S 6508

OSb

O

OSb

OO

O

O-OOC

OHOHHH O- OH

COO-HOHO

3Na+

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

10Ph

osp

ho

ryla

tio

n A

ssay

Kit

sPhosphorylation Assay Kits

Sigma-RBI is pleased to introduce a series of phosphospecific assaykits for the precise quantitation of phosphorylated or non-phospho-rylated cell signaling proteins involved in phosphorylation cascades.Phosphospecific ELISAs (Enzyme-Linked Immuno-sorbent Assay)offer researchers a sensitive, quantitative and economical alternativeto traditional immunoblotting or functional assays.

Assay Platform

The format is a solid phase sandwich ELISA. A capture antibody,specific for a protein, regardless of phosphorylation state, has beencoated onto the wells of the microtiter plate. Following binding ofantigen to the capture antibody, a detection antibody specific forthe phosphorylated or non-phosphorylated protein is added andbinds to the immobilized protein. An anti-rabbit IgG-HRP completesthe four-member sandwich. The reaction is visualized by tetra-methylbenzidene (TMB) substrate and the intensity of the color isdirectly proportional to the concentration of protein present in theoriginal sample. The reaction is read at 450 nm in a microtiter platereader.

Samples

Cell extracts are the samples of choice because the proteins to bemeasured are present in intracellular compartments. Researchersmay use the cell extraction procedure provided in the TechnicalBulletin or utilize the Sigma Mammalian Cell Lysis Kit (Prod.Code MCL-1).

Advantages

Sensitive• Picogram/mL quantities for non-phosphospecific ELISAs • <1.0 unit/mL for phosphospecific ELISAs• 2 to 10 times more sensitive than traditional immunoblotting

Specific• Highly specific for target protein and phosphorylation site• Validated against immunoblotting performed with the same

antigen and antibodies• Validated by peptide competition

Quantitative• Standard curve is run in each assay

Simple• Precoated plates eliminate the coating step• All incubations performed at room temperature• Small sample size – only 10 µL (diluted to a total volume of 100 µL)• Use any microtiter plate reader available on the market• No additional equipment required

Stable• Entire kit is stable in the refrigerator for 12 months• Components are all stored at 2-8°C• Stable liquid chromogen and stop reagents• Two vials of standard extend the lifespan of the kit

Fast• Results in 4 hours

Economical• Reagents may be used for multiple kit runs

Performance Characteristics• Low intra- and inter-assay variability• Linear standard curve• Recovery ranges >93%• Each phosphospecific kit is normalized against non-phospho-

specific ELISA

Figure 1. Bcl-2 [pSer70] ELISA: Peptide Blocking

Concentration of Peptide (µg/mL)

Figure 2. Bcl-2 [pSer70] and Bcl-2 [Total] ELISAs on Paclitaxel Treated Cells

Figure 3. Bcl-2 [pSer70] ELISA; Paclitaxel treated Jurkat and GTL-16 Cells(Normalized for Total Bcl-2)

O.D

. 450

nm

O.D

. 450

nm

Bcl

-2 [

pSe

r70 ]

(U

nit

s/n

g)

Apoptosis/Cell Cycle

CS0420 Phospho-Retinoblastoma [pThr821] ELISA, humanCS0530 Phospho-Bcl-2 [pSer70] ELISA, human

Cytoskeleton/Extracellular Matrix

CS0440 Phospho-Tau [pSer199] ELISA, mouse

Gene Regulation

CS0480 Phospho-STAT1 [pThr701] ELISA, humanCS0550 Phospho-ATF2 [pThr69/pThr71]CS0570 Phospho-CREB [pSer133] ELISA

Nitric Oxide/Cell Stress

CS0630 Phospho-HSP27 [pSer82] ELISA, human

Protein Phosphorylation

CS0460 Phospho-Src [pTyr418] ELISA, humanCS0590 Phospho-c-Met [pTyr1230/pTyr1234/pTyr1235] ELISACS0650 Phospho-MEK1 [pSer218/pSer222] ELISA

Sample Data using Phospho-Bcl-2 [pSer70]ELISA Kit (Prod. Code CS0530)

New Phosphorylation Assay Kits

For a listing of total ELISAs offered by Sigma-RBI, please refer to page 11.

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

11

Cel

l Sig

nal

ing

Ass

ay K

its

APOPTOSIS AND CELL CYCLE

CS0520 Bcl-2 ELISA, human ELISA 96 wells Quantitative ELISA for measurement of Bcl-2 in cell lysates.

CS0530 Phospho-Bcl-2 [pSer70] ELISA Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of Bcl-2 phosphorylated at[pSer70] in cell lysates.

CS0720 Citrate synthase assay kit Enzymatic/Colorimetric 100 test/1mL Colorimetric assay of citrate synthase activity, a mitochondrial matrix 480 tests/well marker.

CS0420 Phospho-Retinoblastoma [pThr821] Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of Rb phosphorylated at [pThr821] ELISA, human in cell lysates.

CS0510 TRAIL ELISA, human ELISA 96 wells Quantitative ELISA for measurement of TRAIL in cell lysates.

CYTOSKELETON AND EXTRACELLULAR MATRIX

ACE100 Cultured cells acellularization kit Cell Fractionation 30 preparations Growth and separation of extracellular matrix secreting cells.

CS0430 Tau ELISA, mouse ELISA 96 wells Quantitative ELISA for measurement of Tau in cell lysates.

CS0440 Phospho-Tau [pSer199] ELISA, mouse Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of Tau phosphorylated at [pSer199]in cell lysates.

GENE REGULATION AND EXPRESSION

CS0540 ATF2 ELISA (total) ELISA 96 wells Quantitative ELISA for measurement of ATF2 in cell lysates.

CS0550 Phospho-ATF2 [pThr69/pThr71] Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of ATF2 phosphorylated at[pThr69/pThr71] in cell lysates.

CS0580 CREB ELISA ELISA 96 wells Quantitative ELISA for measurement of CREB in cell lysates.

CS0570 Phospho-CREB [pSer133] ELISA Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of CREB phosphorylated at[pSer133] in cell lysates.

CS0620 IκBα ELISA ELISA 96 wells Quantitative ELISA for measurement of IκBα in cell lysates.

PEROX1 Peroxisome isolation kit Cell Fractionation 50 g tissue or Isolation of enriched peroxisomal fraction from cells and tissues.20 mL packed cells

CS0470 STAT1 ELISA ELISA 96 wells Quantitative ELISA for measurement of STAT1 in cell lysates.

CS0480 Phospho-STAT1 [pThr701] ELISA Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of STAT1 phosphorylated at[pThr701] in cell lysates.

LIPID SIGNALING

CS0220 Leukotriene B4 (LTB4) EIA EIA 96 wells Competitive emzyme immunoassay (EIA) for the quantitativedetermination of LTB4 in biological samples and lysates.

LYSISO1 Lysosome isolation kit Cell Fractionation 25 g tissue or Isolation of enriched lysosomal fraction from cells and tissues.20 mL packed cells

CS0190 Thromboxane B2 (TXB2) EIA EIA 96 wells Competitive immunoassay (EIA) for the quantitative determination ofTXB2 in biological samples and lysates.

NEUROBIOLOGY

CS0500 Amyloid precursor protein ELISA, human ELISA 96 wells Quantitative ELISA for measurement of APP in cell lysates.

NEUROTRANSMISSION

CS0180 Substance P EIA EIA 96 wells Competitive immunoassay (EIA) for the quantitative determination ofSubstance P in biological samples and lysates.

NITRIC OXIDE AND CELL STRESS

CS0740 Acid phosphatase assay kit Enzymatic/Colorimetric 1,000 in microwell Quantitative acid phosphatase activity measurement in tissues, whole plate cell extracts, column fractions and purified enzyme. Acid phosphatase

can serve as a lysosomal marker.

CS0630 Phospho-HSP27 [pSer82] ELISA, human Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of HSP27 phosphorylated at[pSer82] in cell lysates.

CS0640 HSP27 ELISA, human ELISA 96 wells Quantitative ELISA for measurement of HSP27 in cell lysates.

CS0710 Ogg1 assay kit Enzymatic/Radioactive 180 assays Detection of Ogg1 enzyme activity (DNA repair) in cell lysates.

PROTEIN PHOSPHORYLATION

CS0600 Casein kinase I activity assay kit Enzymatic/Radioactive 50 tests Enzymatic activity of casein kinase-I (CK 1) assayed by radioactivedetection of the phosphorylated peptide substrate.

CS0610 Casein kinase II activity assay kit Enzymatic/Radioactive 70 tests Enzymatic activity of casein kinase-II (CK 2) assayed by radioactivedetection of the phosphorylated peptide substrate.

CS0490 MEK1 assay kit Enzymatic/Immunoblotting 50 reactions Enzymatic activity of MEK1 immunoprecipitated from cell lysates andassayed by immunoblotting of the phosphorylated MEK1 substrate ERK2.

CS0650 Phospho-MEK1 [pSer218/pSer222] Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of MEK1 phosphorylated at ELISA [pSer218/pSer222] in cell lysates.

CS0700 MEK1 ELISA ELISA 96 wells Quantitative ELISA for measurement of MEK1 in cell lysates.

CS0560 c-Met ELISA ELISA 96 wells Quantitative ELISA for measurement of c-Met in cell lysates.

CS0590 Phospho-c-Met [pTyr1230/pTyr1234/ Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of c-Met phosphorylated at pTyr1235] ELISA [pTyr1230/pTyr1234/pTyr1235] in cell lysates.

CS0450 Src ELISA, human ELISA 96 wells Quantitative ELISA for measurement of Src in cell lysates.

CS0460 Phospho-Src [pTyr418] ELISA Phosphospecific ELISA 96 wells Quantitative ELISA for measurement of Src phosphorylated at [pTyr418]in cell lysates.

CS0730 Tyrosine kinase assay kit Enzymatic/Radioactive/ 50 reactions Enzymatic activity measurement of tyrosine kinase immunoprecipitated Dot Blot from cell lysates by radioactive or dot blot detection of the phosphory-

lated poly (Glu, Tyr) substrate.

Prod. Code Name Detection Size Description

New Cell Signaling Assay Kits Available from Sigma-RBI

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

12N

ew P

rod

uct

s

APOPTOSIS AND CELL CYCLE

B 4060 BAFF Receptor/Fc Chimera, human, recombinant,expressed in NSO cells

S 5944 S-15176 difumarate saltMitochondrial permeability transition pore inhibitor;antioxidant.

T 5075 Anti-Tuberin (VV-18) (rabbit)

V 3639 Valinomycin Ready Made*Potassium ionophore, which uncouples oxidative phos-phorylation, induces apoptosis in murine thymocytes,inhibits NGF-induced neuronal differentiation, and antagonizes ET-induced vasoconstriction. *Provided as an ~1 mg/ml solution in DMSO.

CYTOKINES, GROWTH FACTORS AND HORMONES

A 1604 Activin AB, human, recombinant, expressed in CHO cells

A 0729 Monoclonal Anti-Activin B, Clone 146807 (mouse)

A 1729 Activin B, human, recombinant, expressed in CHO cells

A 0604 Monoclonal Anti-Angiopoietin-1, Clone 171718 (mouse)

A 1104 Anti-Angiopoietin-1 (goat)

A 1229 Angiopoietin-3, mouse, recombinant, expressed in NSO cells

A 0854 Monoclonal Anti-Angiopoietin-4, Clone 156215 (mouse)

A 0979 Anti-Angiopoietin-4 (goat)

B 3935 Monoclonal Anti-BMP-8, Clone 158708 (mouse)

C 4866 Cardiogenol C hydrochlorideCardiomyogenesis inducer in embryonic stem cells.

C 2741 Anti-CCR1 (Chemokine Receptor-1) (rabbit)

C 2991 Anti-CCR2 (Chemokine Receptor-2) (rabbit)

C 3241 Anti-CCR7 (Chemokine Receptor-7) (rabbit)

C 8615 CXCL16, human, recombinant, expressed in E. coli

C 1366 Anti-CXCR3 (CXC Chemokine Receptor-3) (rabbit)

C 3116 Anti-CXCR4 (CXC Chemokine Receptor-4) (rabbit)

C 3366 Anti-CXCR5 (CXC Chemokine Receptor-5) (rabbit)

C 8490 CRG-2 (Cytokine Responsive Gene-2), mouse,recombinant, expressed in E. coli

C 8365 CTACK (Cutaneous T-cell-attracting), human,recombinant, expressed in E. coli

E 8780 Epiregulin, mouse, recombinant, expressed in E. coli

E 9530 Erythropoietin, mouse, recombinant, expressed in NSO cells

E 0281 Anti-Estrogen-Related Receptor γ (ERRγ/NR3B3) (rabbit)

E 0406 Anti-Estrogen-Related Receptor α (ERRα/NR3B1) (rabbit)

E 0156 Anti-Estrogen-Related Receptor β (ERRβ/NR3B2) (rabbit)

F 3929 Anti-Follicle-Stimulating Hormone Receptor (rabbit)G 1045 Anti-Glucagon Receptor (rabbit)

G 0920 Anti-Gonadotropin-Releasing Hormone Receptor (rabbit)

G 8919 Anti-Growth Hormone Receptor (goat)H 5163 Hedgehog Interacting Protein, mouse, recombinant,

expressed in NSO cells

I 8783 Anti-Interleukin-19 (goat)I 8908 Anti-Interleukin-20 (goat)

CYTOKINES, GROWTH FACTORS AND HORMONES(continued)

I 8533 Anti-Interleukin-21 (goat)

I 8408 Monoclonal Anti-Interleukin-22, Clone 142938 (mouse)

I 8658 Anti-Interleukin-22 (goat)

J 4145 JervineSonic hedgehog (Shh) pathway inhibitor.

L 6792 Anti-Luteinizing Hormone/ChoriogonadotropinReceptor (rabbit)

M 9193 Anti-Melanocortin-1 Receptor (MC1-R) (rabbit)

M 8693 Melanotan IIMC3-R/MC4-R melanocortin receptor agonist.

N 9411 Nerve Growth Factor Receptor/Fc Chimera, mouse,recombinant, expressed in NSO cells

O 4514 Anti-Orexin Receptor 1 (rabbit)

O 4389 Anti-Oxytocin Receptor (rabbit)

P 6122 Anti-Parathyroid Hormone Receptor 1 (rabbit)

R 2778 RU 28362Glucocorticoid receptor agonist.

S 4944 Monoclonal Anti-Sonic Hedgehog, N-terminal, Clone 171018 (rat)

T 5325 Anti-Thyrotropin (TSH) Receptor (rabbit)

T 2450 Anti-TrkC (goat)

V 5514 Anti-Vasopressin V2 Receptor (rabbit)

CYTOSKELETON AND EXTRACELLULAR MATRIX

A 6354 Anti-Adiponectin (rabbit)

K 0889 Anti-Kinesin 5A (rabbit)

K 1014 Anti-Kinesin 5B (rabbit)

K 0764 Anti-Kinesin 5C (rabbit)

O 3514 Osteopontin, bovine

O 4264 Osteopontin, human, recombinant, expressed in NSO cells

O 3389 Anti-Osteopontin (goat)

P 9371 Monoclonal Anti-Pinch-1, Clone PINCH-N173 (mouse)

P 7372 Anti-Protein Disulfide Isomerase (PDI) (MD-12)(rabbit)

P 7122 Anti-Protein Disulfide Isomerase (PDI) (DL-11)(rabbit)

P 0497 Anti-PMP70 (rabbit)

T 3950 Monoclonal Anti-δ Tubulin, Clone DTU-64 (mouse)

G PROTEINS AND CYCLIC NUCLEOTIDES

A 6604 A-350619Novel, soluble guanylyl cyclase activator. Sold under license from Abbott Laboratories.

R 6153 Monoclonal Anti-RhoE, Clone 4 (mouse)

New Products for Cell Signaling & Neuroscience

New

Pro

du

cts

New Products for Cell Signaling & Neuroscience

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

13

New

Pro

du

cts

GENE REGULATION AND EXPRESSION

A 5854 Monoclonal Anti-AFX (FOXO4), Clone AF3.10 (mouse)

D 6567 DRF 2519Dual PPARα/γ agonist.

F 1304 Monoclonal Anti-FKHRL1 (FOXO3a), Clone FR1 (mouse)

F 1554 Monoclonal Anti-FXR2, Clone A42 (mouse)

H 9163 Anti-HDRP/MITR (rabbit)

D 5567 Anti-dimethyl-Histone H3 [diMe-Lys9] (rabbit)

H 8163 Anti-Histone Deacetylase 5 (NA-16) (rabbit)

H 6663 Monoclonal Anti-Histone Deacetylase 7, Clone HDAC7-97 (mouse)

H 8038 Anti-Histone Deacetylase 8 (rabbit)

D 5692 Anti-dimethyl-Histone H3 [diMe-Lys4] (rabbit)

D 5567 Anti-dimethyl-Histone H3 [diMe-Lys9] (rabbit)

I 9658 Monoclonal Anti-Importin α1, Clone 1A6 (rat)

I 9783 Monoclonal Anti-Importin α3, Clone 3D10 (rat)

I 9908 Monoclonal Anti-Importin α5/7, Clone 2D9 (rat)

M 7568 Anti-MBD2a (Methyl CpG Binding Domain-2a(rabbit)

M 7318 Anti-MBD2a,b (RA-18) (Methyl CpG BindingDomain-2a,b (rabbit)

M 7443 Anti-MeCP2 (rabbit)

M 0944 Anti-Msx1 (rabbit)

N 8786 Anti-Nuclear Pore Complex Proteins, Clone 4 (mouse)

P 0748 Monoclonal Anti-PRMT2, Clone PRMT2-34 (mouse)

R 6153 Monoclonal Anti-RhoE, Clone 4 (mouse)

R 8903 Monoclonal Anti-hnRNP-K/J, Clone 3C2 (mouse)

R 6278 Monoclonal Anti-hnRNP-U, Clone 3G6 (mouse)

S 1945 Anti-SF1 (Anti-Splicing Factor-1) (rabbit)

S 2070 Anti-SF4 (Anti-Splicing Factor 4) (rabbit)

S 2195 Anti-SFRS1/9 (Anti-Splicing Factor arginine/serinerich 1/9) (rabbit)

S 2320 Anti-SFRS2 (Anti-Splicing Factor arginine/serine rich 2)(rabbit)

S 2445 Anti-SFRS3 (Anti-Splicing Factor arginine/serine rich 3)(rabbit)

S 2570 Anti-SFRS4 (Anti-Splicing Factor arginine/sering rich 4)(rabbit)

S 2695 Anti-SFRS5 (Anti-Splicing Factor arginine/serine rich 5)(rabbit)

S 2820 Anti-SFRS6 (Anti-Splicing Factor arginine/serine rich 6)(rabbit)

S 4070 Anti-SFRS10 (Anti-Splicing Factor arginine/serinerich 10) (rabbit)

S 2945 Anti-SFRS12 (Anti-Splicing Factor arginine/serinerich 12 (rabbit)

S 3070 Anti-SFRS14 (Anti-Splicing Factor arginine/serinerich 14) (rabbit)

S 3195 Anti-SR-A1 (Anti-Splicing Factor SR-A1) (rabbit)

S 3320 Anti-SRRM1 (Anti-Serine/Arginine RepetitiveMatrix-1) (rabbit)

IMMUNE CELL SIGNALING AND BLOOD

S 1820 Ser-Phe-Leu-Leu-Arg-Asn-amide (SFLLRN-NH2)Thrombin receptor activating peptide.

A 1354 APRIL (A Proliferation-inducing Ligand), human,recombinant, expressed in NSO cells

M 8067 Anti-Mouse IgG2b (heavy chain specific) (goat)

ION CHANNELS

A 3979 Anti-Na+/K+ ATPase β2 (rabbit)

A 5229 Agitoxin-1, recombinant, expressed in E. coliPotent blocker of Shaker voltage-gated K+ channels.

C 1116 Anti-Chloride Channel CLC-5 (Clcn5) (rabbit)

C 2366 Anti-K+/Cl- Cotransporter (KCC2) (rabbit)

C 2491 Anti-Calsequestrin (cardiac) (rabbit)

C 4616 Anti-Calcium Channel CaV3.3 (α1I) (rabbit)

C 5238 Chlorotoxin, recombinant, expressed in E. coliBlocker of small conductance Cl- channels.

E 9904 Ergtoxin, recombinant, expressed in E. coliSpecific ERG (ether-a-go-go) K+ channel blocker.

G 2795 ω-Grammotoxin SIA, from Grammostola spatulata(Tarantula)Novel blocker of CaV2.1 (P-type) and CaV2.2 (N-type)voltage-gated Ca2+ channels.

K 1139 Monoclonal Anti-Kv2.1 α-subunit, Clone D4/11

K 1264 Monoclonal Anti-Potassium Kv1.4 α-subunit, Clone K13/31

K 1389 Monoclonal Anti-Potassium Kv1.1 α-subunit, Clone K20/78

K 1514 KurtoxinT-type Ca2+ channel blocker; scorpion toxin.

N 0287 NNC 55-0396Selective T-type Ca2+ channel blocker.

P 0248 PNU-37883ASelective Kir 6.1/SUR2B channel blocker.

P 3495 Phrixotoxin-2, from Phrixotrichus auratus (Tarantula)Specific, reversible KV4.2 and KV4.3 K+ channel blocker.

P 4122 Monoclonal Anti-PMCA4b (Ca2+ ATPase), Clone JA3(mouse)

P 4497 Anti-Potassium Channel KV1.3 (extracellular) (rabbit)

P 9497 Anti-Potassium Channel hKV11.1 (HERG) (rabbit)

P 9872 Psora-4Potent KV1.3 K+ channel blocker.

P 1373 Anti-Voltage Gated Potassium Channel, Kv2.2 Subunit(rabbit)

T 6700 TRAM-34Potent intermediate-conductance Ca2+ activated K+

channel blocker.

V 3639 Valinomycin Ready Made*Potassium ionophore, which uncouples oxidative phos-phorylation, induces apoptosis in murine thymocytes,inhibits NGF-induced neuronal differentiation, and antagonizes ET-induced vasoconstriction. *Provided as an ~1 mg/ml solution in DMSO.

Z 0127 Zatebradine hydrochlorideHCN channel blocker; blocker of neuronal Ih, relatedcardiac If channels and ATP-sensitive Kir channels.

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

14N

ew P

rod

uct

s

LIPID SIGNALING

A 6354 Anti-Adiponectin (rabbit)

A 0354 Anti-Adipocyte Complement Related Protein of 30 kDa (Acrp30) (rabbit)

B 8685 BM 15.766 sulfate∆7-dehydrocholesterol reductase inhibitor.

C 3491 Anti-Cysteinyl Leukotriene Receptor 1 (rabbit)

D 7692 Diethylumbelliferyl phosphate (UBP; DEUP)Selective and potent cholesterol esterase inhibitor; blockssteroidogenesis by preventing cholesterol transport intomitochondria of steroidogenic cells.

G 2295 GlimepirideSulfonylurea used in treatment of Type 2 diabetes.

L 7042 Anti-Leukotriene B4 Receptor BLT2 (rabbit)

P 0247 L-NASPACompetive mammalian lysophosphatitic acid (LPA)receptor agonist.

R 0279 Ro 23-9358Non-pancreatic sPLA2 inhibitor.

R 9028 RepaglinideNon-sulfonylurea oral hypoglycemic agent.

T 6575 TOFACell-permeable, potent, reversible and competitive acetyl-CoA carboxylase inhibitor.

MULTI-DRUG RESISTANCE

B 7185 Anti-Breast Cancer Resistance Protein (rabbit)

NEUROBIOLOGY

N 9286 Notch-1/Fc Chimera, rat, recombinant, expressed in NSO cells

NEUROTRANSMISSION

A 0229 β2-Adrenergic Receptor PreparationFrozen aliquot of membranes, suspended in 50 mM tris-HCl containing 12 mM MgCl2 and 2 mM EDTA, pH 7.4 at20° C.

A 4104 Anti-A1 Adenosine Receptor (rabbit)

A 4604 Anti-α1C Adrenergic Receptor (rabbit)

A 4354 Anti-α1D Adrenergic Receptor (rabbit)

A 4729 Anti-β2 Adrenergic Receptor (rabbit)

A 4854 Anti-β3 Adrenergic Receptor (rabbit)

A 2729 AmisulprideHighly selective D2/D3 dopamine receptor antagonist;atypical antipsychotic.

A 8978 Monoclonal Anti-β-Amyloid [13-28], Clone BAM90.1(mouse)

B 4310 BIM 23056Selective sst5 somatostatin receptor antagonist.

B 5560 Anti-B1 Bradykinin Receptor (rabbit)

NEUROTRANSMISSION (continued)

B 5685 Anti-B2 Bradykinin Receptor (rabbit)

C 2490 CYN 154806Potent, selective sst2 somatostatin receptor antagonist.

C 2866 Anti-CB1 Cannabinoid Receptor (rabbit)

D 6692 Anti-D1 Dopamine Receptor (rabbit)

D 6817 Anti-D4 Dopamine Receptor (rabbit)

E 9780 Anti-Endothelin A Receptor (rabbit)

E 9905 Anti-Endothelin B Receptor (rabbit)

F 4429 FAUC 213Highly selective D4 dopamine receptor antagonist.

F 3804 Anti-Frizzled-1 (FZD-1) (rabbit)

F 3304 Anti-Frizzled-2 (FZD-2) (rabbit)

F 3179 Anti-Frizzled-3 (FZD-3) (rabbit)

F 3429 Anti-Frizzled-4 (FZD-4) (rabbit)

F 3679 Anti-Frizzled-7 (FZD-7) (rabbit)

F 3554 Anti-Frizzled-10 (FZD-10) (rabbit)

G 0420 Anti-Glycine Receptor α3 (rabbit)

G 1295 Anti-GABAB Receptor 1 (rabbit)

G 0795 Anti-Galanin Receptor (GalR1) (rabbit)

G 0670 Anti-Galanin Receptor (GalR3) (rabbit)

G 1420 Anti-Glutamate Receptor 1, Metabotropic (mGluR1)(rabbit)

G 1545 Anti-Glutamate Receptor 3, Metabotropic (mGluR3)(rabbit)

G 1670 Anti-Glutamate Receptor 4, Metabotropic (mGluR4)(rabbit)

G 1795 Anti-Glutamate Receptor 6, Metabotropic (mGluR6)(rabbit)

G 1920 Anti-Glutamate Receptor 7, Metabotropic (mGluR7)(rabbit)

G 2045 Anti-Glutamate Receptor 8, Metabotropic (mGluR8)(rabbit)

G 7669 Anti-Glutamate Receptor 6/7, Kainate (GluR 6/7) (rabbit)

H 6913 Anti-H1 Histamine Receptor (rabbit)

H 7038 Anti-H3 Histamine Receptor (rabbit)

L 6292 Lisinopril (MK-521)Angiotensin converting enzyme (ACE) inhibitor.

M 3443 MSX-2Selective A2A adenosine receptor antagonist.

M 3568 MSX-3Selective water-soluble A2A adenosine receptor antagonistprodrug.

N 9911 Neuropeptide W-30 (human)Food intake-regulating peptide; GPR7/GPR8 ligand.

O 3639 Ondansetron hydrochloride5-HT3 Serotonin receptor antagonist.

O 3764 OxcarbazepineAnticonvulsant; antineuralgic; inhibits veratrine-inducedtransmitter release.

P 1123 PD 81,723Allosteric enhancer of A1 adenosine receptors.

P 1372 Paroxetine maleateSelective serotonin reuptake inhibitor (SSRI); antidepressant.

P 5622 Anti-Pen-2 (rabbit)

New Products for Cell Signaling & Neuroscience

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

15

New

Pro

du

cts

New Products for Cell Signaling & Neuroscience

NEUROTRANSMISSION (continued)

P 6747 Anti-P2Y1 Purinergic Receptor (rabbit)

P 6497 Anti-P2Y4 Purinergic Receptor (rabbit)

P 6247 Anti-P2Y10 Purinergic Receptor (rabbit)

S 8194 Salicylidene salicylhydrazidePotent and selective α2β1γ1δ GABAA receptor antagonist.

S 0195 Anti-5-HT4 Serotonin Receptor (rabbit)

S 0445 Anti-5-HT2B Serotonin Receptor (rabbit)

S 3445 SM-19712Potent, selective, nonpeptide endothelin convertingenzyme (ECE) inhibitor.

S 9819 Anti-Smoothened Drosophila Homolog (SMOH) (rabbit)

S 0695 Anti-Somatostatin Receptor Type 2

T 8949 TelmisartanNon-peptide AT1 angiotensin receptor antagonist.

U 4258 UCM 171975-HT3 Serotonin receptor antagonist.

NITRIC OXIDE AND CELL STRESS

A 6229 Monoclonal Anti-AKR1 C3, Clone NP6.G6.A6 (mouse)

P 1247 Anti-Peroxiredoxin 3 (PRDX3) (rabbit)

T 1075 Anti-Tal (CS-15) (rabbit)

T 1200 Anti-Tal (FQ-17) (rabbit)

PLANT BIOTECHNOLOGY

M 8318 Anti-AtMPK3 (Arabidopsis thaliana MPK3) (rabbit)

A 6979 Anti-AtMPK4 (Arabidopsis thaliana MPK4) (rabbit)

A 7104 Anti-AtMPK6 (Arabidopsis thaliana MPK6) (rabbit)

C 8115 CoronatinAntibiotic; polyketide phytotoxin produced by severalmembers of the Pseudomonas syringae group ofpathovars; known to induce hypertrophy and chlorosis;inhibits root elongation; stimulates ethylene production.

PROTEIN PHOSPHORYLATION

C 6115 Anti-CIN85 (rabbit)

D 9192 4,5-Dimethoxy-6-nitrobenzaldehyde (DMNB)DNA-dependent protein kinase (DNA-PK) inhibitor.

J3020 Anti-JAB1 (rabbit)

N 2162 NH125Novel plant histidine kinase inhibitor and selective mam-malian eurkaryotic elongation factor-2 kinase (eEF-2K)inhibitor.

P 5747 Monoclonal Anti-Phosphoserine, Clone PSR-45 (mouse)

P 5872 Monoclonal Anti-Phosphotyrosine, Clone PT-66 (mouse)

R 3028 Anti-RALT/ MIG-6 (PE-16) (rabbit)

R 6028 Anti-ROCK-1 (rabbit)

R 8653 Anti-ROCK-2 (rabbit)

R 9153 Anti-Rhodopsin (rabbit)

S 1195 ST638Protein tyrosine kinase inhibitor.

T 8325 TDZD-8Selective glycogen synthase kinase-3 (GSK-3) inhibitor.

T 2450 Anti-TrkC (goat)

FAM-FLICA Caspase Detection Kits

CS0280 Caspase 1 Fluorescein (FLICA) Assay (FAM-VAD-FMK)CS0290 Caspase 2 Fluorescein (FLICA) Assay (FAM-VDVAD-FMK)CS0300 Caspase 9 Fluorescein (FLICA) Assay (FAM-LEHD-FMK)CS0310 Caspase 10 Fluorescein (FLICA) Assay (FAM-AEVD-FMK)CS0320 Caspase 13 Fluorescein (FLICA) Assay (FAM-LEED-FMK)

Caspase detection kits available from Sigma-RBI use FluorochromeInhibitor of Caspases (FLICA) reagent (FAM-XXXD-FMK), whichconsists of a fluorescent-labeled enzyme that is a potent inhibitorof caspases. FLICAs are cell permeable and non-cytotoxic. Insidethe cell, the FLICA covalently binds to the active caspase het-erodimer, inhibiting further enzymatic caspase activity. The FLICAcovalently coupled to the enzyme is retained within the cell, whilethe unbound FLICA reagent diffuses out of the cell and is washedaway. The remaining green fluorescent signal is a direct measureof the number of active caspase molecules present in the cell atthe time the reagent was added.

Detection: Fluorometry, fluorescence microscopy, flowcytometry

Species specificity: MammalianSize: Sufficient for 100 tests

Features and Benefits:• The reagent is cell permeable and non-cytotoxic• Fluorescent signal detects active caspases• Results quantitative by flow cytometry

Sample Results:• Non-induced Jurkat cells were treated with DMSO (A) or

induced with camptothecin for 3 hours (B)• Cells were labeled with FAM-VAD-FMK for 1 hour and washed• Caspase activity was detected using a flow cytometer• Induced cells (B) show 2 peaks: caspase-negative cells occur to

the left of the M1 region (unlabeled cells); caspase-positive cellslay within the M1 region (cells were labeled with FLICA).

Flow Cytometry Detection of Caspase 1

(A) DMSO (B) Camptothecin

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

16Monoclonal Anti-AKR1C3 (Aldo-KetoReductase 1C3)

Prod. Code: A 6229Clone Name: NP6.G6.A6, developed in mouseProduct Form: Purified mouse immunoglobulinImmunogen: human AKR1C3 protein [1]Isotype: IgG1Species Cross Reactivity: human

Aldo-keto reductases (AKRs) are enzymes that perform oxido-reduction on natural and foreign substrates and play a centralrole in the metabolism of natural products, drugs, xenobioticsand carcinogens. The AKR1 (aldo-keto reductases 1) family is thelargest among the 14 AKR families and includes the aldose reduc-tases, aldehyde reductases, hydroxysteroid dehydrogenases, andsteroid 5 β-reductases [2]. In humans, the four isoforms AKR1C1-4 catalyze the reduction of the androgen 5α-dihydrotestosterone(DHT) into inactive 3β or 3α androstanediol (3α/β-diol). In vitro,these enzymes also display 3α[17β]-hydroxysteroid oxidase activityusing 3α-diol as a substrate [3]. AKR1Cs are expressed in manytissues and their expression is dramatically increased in non-smallcell lung carcinoma [4]. Due to their product profile and discretetissue localization, AKR1Cs may regulate the level of active andro-gens, estrogens and progestins in target tissues [5].

Applications: Immunoblotting (~38 kDa), ELISA and immuno-histochemistry

References1. Lin, H.K., et al., Characterization of a monoclonal antibody for human

aldo-keto reductase AKR1C3 (type 2 3α-hydroxysteroid dehydrogenase/type 5 17β-hydroxysteroid dehydrogenase); immunohistochemical detec-tion in breast and prostate. Steroids, 69, 795-801 (2004).

2. Hyndman, D., et al., The aldo-keto reductase superfamily homepage.Chem. Biol. Interact., 143-144, 621-631 (2003).

3. Steckelbroeck, S., et al., Human cytosolic 3α-hydroxysteroid dehydro-genases of the aldo-keto reductase superfamily display significant 3β-hydroxysteroid dehydrogenase activity: implications for steroid hormonemetabolism and action. J. Biol. Chem., 279, 10784-10795 (2004).

4. Palackal, N.T., et al., Activation of polycyclic aromatic hydrocarbon trans-dihydrodiol proximate carcinogens by human aldo-keto reductase (AKR1C)enzymes and their functional overexpression in human lung carcinoma(A549) cells. J. Biol. Chem., 277, 24799-24808 (2002).

5. Penning, T.M., et al., Human 3α-hydroxysteroid dehydrogenase isoforms(AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plas-ticity and tissue distribution reveals roles in the inactivation and formationof male and female sex hormones. Biochem. J., 351, 67-77 (2000).

Cardiogenol C hydrochloride:Cardiomyogenesis inducer

Potent, cell permeable inducer of ESC(embryonic stem cell) differentiation incardiomyocytes displaying an EC50 of100 nM.

References1. Wu, X., et. al., Small molecules that induce cardiomyogenesis in embryonic

stem cells. J. Am. Chem. Soc., 126, 1590-1591 (2004).

New

Pro

du

ct H

igh

ligh

ts

N

N

NH

HO

NH

H3CO HCl

Prod. Code C 4866

1 2

M.W.

50 —

35 —

30 —

Immunoblot:Total cell extract from A549 cells wereseparated by SDS-PAGE and probed withMonoclonal Anti-AKR1C3, CloneNP6.G6.A6 (Prod. Code A 6229) (Lane 1) orwithout primary antibody (Lane 2) andfurther incubated with goat anti-mouseIgG, alkaline phosphatase conjugate(Prod. Code A 2179).

Monoclonal Anti-β-Amyloid [13-28]

Prod. Code: A 8978Clone Name: BAM90.1, developed in mouseProduct Form: Purified mouse immunoglobulinImmunogen: synthetic peptide corresponding to amino acids

13-28 of human β-amyloidIsotype: IgG1Species Cross Reactivity: humanThe antibody epitope resides within amino acids 20-23.

The β-amyloid precursor protein (APP) is cleaved sequentially bythe proteolytic enzymes β-secretase and γ-secretase to produce β-amyloid (Aβ) peptides with the Aβ1-42 and the Aβ1-40 formsbeing the most prevalent. Extracellular accumulation of Aβ leadsto formation of aggregates, fibrils and eventually amyloiddeposits called neuritic plaques, a hallmark of Alzheimer’s dis-ease (AD) [1]. Of the many proposed mechanisms of AD proteintoxicity, one may be through calcium-mediated neurotoxicity. Aβpeptides can increase calcium influx through voltage-gated N- and L-type calcium channels, reduce the magnesium blockadeof NMDA receptors to allow increased calcium influx, and canform a cation-selective ion channel after their incorporation intothe cell membrane [2-3]. Thus, Aβ peptides may elicit toxiceffects prior to fibril formation. Aβ peptides also have beenfound to exhibit superoxidase dismutase activity, thus producinghydrogen peroxidase that may be responsible for neurotoxicity [4].

Applications: ELISA, immunoblotting, immunoprecipitation,immunohistochemistry and in vivo sequestrationof endogenous plasma human β-Amyloid peptide(1-40).

References1. Law, A., et al., Say NO to Alzheimer's disease: the putative links between

nitric oxide and dementia of the Alzheimer's type. Brain Res. Rev., 35, 73-96 (2001).

2. Pearson, H.A., in Alzheimer’s Disease: Methods and Protocols, Hooper,N.M. (Ed.) pp. 113-138, Humana Press, NJ (2000).

3. Zhu, Y.J., et al., Fresh and nonfibrillar amyloid beta protein (1-40) inducesrapid cellular degeneration in aged human fibroblasts: evidence forAbetaP-channel-mediated cellular toxicity. FASEB J., 14, 1244-1254 (2000).

4. Veurink, G., et al., Genetics, lifestyle and the roles of amyloid beta andoxidative stress in Alzheimer's disease. Ann. Hum. Biol., 30, 639-667 (2003).

Related Products

Product Name Host Clone Prod. Code

Monoclonal Anti-β-Amyloid [1-17] Mouse 6E10 A 1474

Monoclonal Anti-β-Amyloid [17-24] Mouse 4G8 A 1349

Monoclonal Anti-β-Amyloid Protein Mouse BAM-10 A 5213

Anti-Amyloid Peptide β, Rabbit A 1976Cleavage Site 42

Anti-Amyloid Peptide β, Rabbit A 2101Cleavage Site 43

Anti-β-Amyloid Protein (1-40) Rabbit A 8326

BIM 23056: Selective sst5 somatostatin receptorantagonist

Prod. Code B 4310

Antagonizes somatostatin-induced intracellular calcium increasein CHO cells expressing human sst5 somatostatin receptors (pKB ~8; Ki = 5.7 nM).

References1. Nunn, C., et al., Pharmacological characterization of the goldfish somato-

statin sst5 receptor. Eur. J. Pharmacol., 436, 173-186 (2002).2. Wilkinson, G.F., et al., Potent antagonism by BIM-23056 at the human

recombinant somatostatin sst5 receptor. Br. J. Pharmacol., 118, 445-447(1996).

3. Raynor, K., et al., Cloned somatostatin receptors: identification of subtype-selective peptides and demonstration of high affinity binding of linearpeptides. Mol. Pharmacol., 43, 838-844 (1993).

D-Phe-Phe-Tyr-D-Trp-Lys-Val-Phe-D-Nal-NH2

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

17

New

Pro

du

ct H

igh

ligh

ts

Available First from Sigma-RBI!

Ondansetron hydrochloride and UCM-17197:Potent, selective and orally active 5-HT3 serotoninreceptor antagonists

Prod. Code O 3639 Prod. Code U 4258Antagonists of the 5-HT3serotonin receptor havebeen used clinically toreduce emesis associatedwith cancer chemo-therapy and are under

investigation for the treatment of irritable bowel syndrome,visceral pain and fibromyalgia [1,2].

Ondansetron (GR-38032F, Prod. Code O 3639), which has beenused clinically since 1991 to prevent nausea and vomitinginduced by cancer chemotherapy, radiation therapy, anesthesiaand surgery, displays a Ki of 0.77 nM at 5-HT3 serotonin recep-tors and >1,000 nM at 5-HT4 serotonin receptors as assessed byradioligand binding assay in rat cerebral cortex membranes [2,3].Current evidence also indicates that ondansetron may reducealcohol consumption in moderate alcohol abusers [2].

UCM-17197 (Prod. Code U 4258) is a structurally novel benzimi-dazole derivative that exhibits somewhat greater potency andselectivity for 5-HT3 serotonin receptors (Ki = 0.13 nM at 5-HT3serotonin receptors, Ki >10,000 nM at 5-HT4 serotonin receptorsassessed by radioligand binding assay in rat cerebral cortexmembranes) than ondansetron [3]. UCM-17197 also demon-strates slightly more pronounced pharmacological activity in iso-lated guinea pig ileum (ED50 of 17 µM for UCM-17197 vs. 4.3 µM for ondansetron) [3]. Both compounds dose-dependentlyinhibit serotonin-induced bradycardia in anesthesized rats andreduce gastric emptying and diarrhea in mice following intraperi-toneal or oral administration [4].

Ondansetron, a well-known and widely used clinical drug, andUCM-17197 should serve as critical pharmacological tools infurther elucidating the function of 5-HT3 serotonin receptors invarious disease models.

References1. Balfour, J.A., et al., Alosetron. Drugs, 59, 511-518 (2000).2. Costal, B. and Naylor, R.J., 5-HT3 receptors. Curr. Drug Targets CNS Neurol.

Disord., 3, 27-37 (2004).3. Lopez-Rodriguez, M.L., et al., Benzimidazole derivatives. 2. Synthesis and

structure-activity relationships of new azabicyclic benzimidazole-4-car-boxylic acid derivatives with affinity for serotoninergic 5-HT(3) receptors. J. Med. Chem., 42, 5020-5028 (1999).

4. Pascual, D., et al., New benzimidazole derivatives: selective and orallyactive 5-HT3 receptor antagonists. Eur. J. Pharmacol., 462, 99-107 (2003).

HClN

ON N

CH3

H3C

N

HN O

NH

N

Cl

Anti-Apolipoprotein J (Clusterin)

Prod. Code: A 2229Product Form: Developed in goat, affinity isolated antibodyImmunogen: Apolipoprotein Type J isolated from human plasma Species Cross Reactivity: human

Anti-Apolipoprotein J (Apo J, Clusterin) recognizes humanapolipoprotein J while displaying negligible cross-reactivity withType A-I, A-II, B, C-I, C-II, C-III and E apolipoproteins. Apo J is aubiquitously expressed secreted glycoprotein that has been impli-cated in several physiological and disease processes fromAlzheimer’s disease to cancer. It appears to be involved in cellproliferation and differentiation, but its role in cell survival, celldeath and neoplastic transformation remains unclear [1-3]. InAlzheimer’s disease, Apo J (in conjunction with apolipoprotein E)may have profound effects on the onset of amyloid-β deposition,as well as the local toxicity associated with amyloid-β deposits[4]. Apo J is being considered as a possible cerebrospinal fluidmarker for the disease [5].

Applications: Immunoblotting, immunohistochemistry, ELISA

References1. Scaltriti, M., et al., Clusterin (SGP-2, Apo J) expression is downregulated in

low- and high-grade human prostate cancer. Int. J. Cancer, 108, 23-30 (2004).2. Thomas-Tikhonenko A., et al., Myc-transformed epithelial cells down-regu-

late clusterin, which inhibits their growth in vitro and carcinogenesis invivo. Cancer Res. 64, 3126-3136 (2004).

3. Trougakos, I.P, et al., Silencing expression of the clusterin/apolipoprotein Jgene in human cancer cells using small interfering RNA induces sponta-neous apoptosis, reduced growth ability, and cell sensitization to genotox-ic and oxidative stress. Cancer Res., 64, 1834-1842 (2004).

4. Holtzman, D.M., In vivo effects of ApoE and clusterin on amyloid-betametabolism and neuropathology. J. Mol. Neurosci., 23, 247-254. (2004).

5. Puchades, M., et al., Proteomic studies of potential cerebrospinal fluid pro-tein markers for Alzheimer's disease. Brain Res. Mol. Brain Res., 118,140-146 (2003).

Related Products

Product Name Host Clone Prod. Code

Monoclonal Anti-Apolipoprotein E Mouse E6D7 A 8599

Paroxetine maleate: Potent, selective serotoninreuptake inhibitor (SSRI); antidepressantProd. Code P 1372

Binds with high affinity to the serotonintransporter (SERT) displaying a Ki of0.05 nM in rat cortical membranes.

References1. Bourin, M., et al., Paroxetine: a review. CNS Drug Rev., 7, 25-47 (2001).2. Owens, M.J., Neurotransmitter receptor and transporter binding profile of

antidepressants and their metabolites. J. Pharmacol. Exp. Ther., 283, 1305-1322 (1997).

Additional SSRI Inhibitors

Product Name Prod. Code

Citalopram hydrobromide C 7861

Fluoxetine hydrochloride F-132

Fluvoxamine maleate F 2802

NH

F

O

O

O

COOH

COOH

CYN 154806: Potent and selective sst2somatostatin receptor antagonist

Prod. Code C 2490

CYN 154806 binds to sst2 somatostatin receptors with nM affini-ty (Ki = 0.2 nM; pKd = 8.14-8.89 nM), which is 40- to 4,500-foldhigher than its binding to sst1, sst3 and sst4 somatostatin recep-tors. The pIC50 values are 8.6, 5.4, 6.1, and 6.5 for humanrecombinant sst2, sst3, sst4, and sst5 receptors, respectively.

References1. Nunn, C., et al., Functional characterisation of the putative somatostatin

sst2 receptor antagonist CYN 154806. Naunyn Schmiedebergs Arch.Pharmacol., 367, 1-9 (2003).

2. Dal Monte, M., et al., Somatostatin inhibits potassium-evoked glutamaterelease by activation of the sst(2) somatostatin receptor in the mouseretina. Naunyn Schmiedebergs Arch. Pharmacol., 367, 188-192 (2003).

Additional sst2 Somatostatin Receptor Agonists

Product Name Prod. Code

BIM-2307 trifluoroacetate salt B 0434

Seglitide S 1316

Ac-(4-Nitro)Phe-D-Cys-Tyr-D-Trp-Lys-Thr-Cys-D-Tyr-NH2

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

18TRAM-34: Potent intermediate-conductance Ca2+-activated K+ channel blocker

Prod. Code T 6700Used to prevent vascular restenosis followingballoon angioplasty. Displays a 100-foldselectivity (Kd = 20 nM) for IKCa1 channelsover other K+ channels (Charybdotoxin, Kd = 5 nM, Clotrimazole, Kd = 70 nM;Nitrendipine Kd = 900 nM).

References1. Wulff, J. et al., Design of a potent and selective inhibitor of the inter-

mediate-conductance Ca2+-activated K+ channel, IKCa1: a potentialimmunosuppressant. Proc. Natl. Acad. Sci. USA, 97, 8151-8156 (2000).

2. Chandy, K.G., et al., K+ channels as targets for specific immunomodula-tion. Trends Pharmacol. Sci., 25, 280-289 (2004).

Related Products

Product Name Descriptor Prod. Code

Charybdotoxin Ca2+-activated K+ channel inhibitor C 5856

NS309 Intermediate-conductance N 8161Ca2+-activated K+ channel activator

Anti-Potassium Channel KCa3.1 (Intermediate-conductance P 4997Ca2+-activated K+ Channel 4) (rabbit)

PD 81,723: Allosteric enhancer of agonistbinding to A1 adenosine receptors

Prod. Code P 1123PD 81,723 (30 µM) decreases the EC50value for adenosine to prolong the S-Hinterval by nine-fold in guinea pig isolatedheart; 3 mg/kg enhances the neuro-protective and anticonvulsant effects ofadenosine following the induction of hyper-glycemic ischemia in rats.

References1. Kollias-Baker, C., et al., Allosteric enhancer PD 81,723 acts by novel mecha-

nism to potentiate cardiac actions of adenosine. Circ. Res., 75, 961-971 (1994).2. Meno, J.R., et al., Hippocampal injury and neurobehavioral deficits are

improved by PD 81,723 following hyperglycemic cerebral ischemia. Exp.Neurol., 183, 188-196 (2003).

3. Janusz, C.A. and Berman, R.F., The adenosine binding enhancer, PD 81,723,inhibits epileptiform bursting in the hippocampal brain slice. Brain Res.,619, 131-136 (1993).

®

®

New

Pro

du

ct H

igh

ligh

ts

NN

Ph

PhCl

S

H3C

H3C NH2

OCF3

Anti-Frizzled (FZD) Antibodies

Prod. Code: See table belowProduct Form: Developed in rabbit, affinity isolated antibodiesImmunogen: Synthetic peptidesSpecies Cross Reactivity: human

Wnt-Frizzled signaling is an essential component of early devel-opment regulating cell fate, polarity, differentiation, migration,formation of neural synapses and the regulation of proliferation.Frizzled proteins are GPCRs for secreted Wnt proteins, as well asother ligands. They have been identified in a range of animalspecies, from sponges to humans [1,2]. Wnt1 is an oncogenethat causes mouse mammary tumorigenesis. Defective Wnt sig-naling plays major roles in diseases such as cancer (for example,colorectal cancer andmelanomas) and osteo-porosis [3]. Therefore, Wntsignal transduction compo-nents represent promisingtargets in the search fornovel cancer therapeuticagents.

Applications:Immunohistochemistry

References1. Huang, H.C. and Klein, P.S., The

Frizzled family: receptors for mul-tiple signal transduction pathways. Genome Biol., 5, 234 (2004).

2. Vincan, E., Frizzled/WNT signalling: the insidious promoter of tumourgrowth and progression. Front. Biosci., 9, 1023-1034 (2004).

3. Wang, H.Y. and Malbon, C.C., Wnt-frizzled signaling to G-protein-coupledeffectors. Cell Mol. Life Sci., 61, 69-75 (2004).

New Frizzled Antibodies

Product Name Host Prod. Code

Anti-Frizzled-1 (FZD-1) Rabbit F 3804

Anti-Frizzled-2 (FZD-2) Rabbit F 3304

Anti-Frizzled-3 (FZD-3) Rabbit F 3179

Anti-Frizzled-4 (FZD-4) Rabbit F 3429

Anti-Frizzled-7 (FZD-7) Rabbit F 3679

Anti-Frizzled-10 (FZD-10) Rabbit F 3554

Immunochemical staining of fetal kid-ney using Rabbit Anti-Frizzled-2 (Prod.Code F 3304).

Monoclonal Anti-Potassium ChannelKCNK9 (TASK-3)Prod. Code: K 0514Clone Name: KCN, developed in mouseProduct Form: Purified mouse immunoglobulin Immunogen: Synthetic peptide corresponding to amino acids

360-374 in the C-terminus of human KCNK9Isotype: IgG2bSpecies Cross Reactivity: human

KCNK9 or TASK-3 (TWIK-related Acid sensitive K+ channel,K2P9.1) is a member of the potassium channel family of proteinsthat contain two-pore domains and four transmembranedomains. These channels are characterized as leaky K+ channelsthat are sensitive to changes in the extracellular pH. The physio-logical functions of TASK channels are largely unknown. It hasbeen proposed that they may be involved in the regulation ofbreathing, aldosterone secretion and anesthetic-mediatedneuronal activity. [1-4].

Applications: Immunoblotting, immunocytochemistry, ELISA

References1. Kim, Y., et al., TASK-3, a new member of the tandem pore K(+) channel

family. J. Biol. Chem., 275, 9340-9347 (2000).2. Talley, E.M., et al., Modulation of TASK-1 (Kcnk3) and TASK-3 (Kcnk9)

potassium channels: volatile anesthetics and neurotransmitters share amolecular site of action. J. Biol. Chem., 277, 17733-17742 (2002).

3. Talley, E.M., et al., CNS distribution of members of the two-pore-domain(KCNK) potassium channel family. J. Neurosci., 21, 7491-7505 (2001).

4. Pei, L., Oncogenic potential of TASK3 (Kcnk9) depends on K+ channel-function. Proc. Natl. Acad. Sci. USA, 100, 7803-7807 (2003).

Related Products

Product Name Host Prod. Code

Anti-Potassium Channel TASK-1 (KCNK3) Rabbit P 0981

Anti-Potassium Channel TASK-2 (KCNK5) Rabbit P 1106

Anti-Potassium Channel K2P9.1 (TASK-3) (KCNK9) Rabbit P 5247

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

19

Prod. Code T 2825

Cell permeable, potent PARP inhibitor;IC50 of 450 nM versus recombinant bovinePARP-1; shown to be neuroprotectiveagainst oxygen-glucose deprivation injuryin vitro in cultured murine cortical cells(IC50 = 150 nM) and in vivo in rat tran-sient focal ischemia model (3 mg/kg ip).

References1. Chiarugi A, et al., Novel isoquinolinone-derived inhibitors of poly(ADP-

ribose) polymerase-1: pharmacological characterization and neuroprotec-tive effects in an in vitro model of cerebral ischemia. J. Pharmacol. Exp.Ther., 305, 943-949 (2003).

2. Pellicciari, R., et al., Towards new neuroprotective agents: design and syn-thesis of 4H-thieno[2,3-c] isoquinolin-5-one derivatives as potent PARP-1inhibitors. Farmaco., 58, 851-858 (2003).

Additional PARP Inhibitors

Product Name Prod. Code

3-Aminobenzamide A 0788

4-Amino-1,8-naphthalimide A 0966

DPQ D 5314

1,5-Isoquinolinediol I-138

PJ-34 P 4365

6(5H)-Phenanthridinone P 8852

TIQ-A: Poly(ADP-Ribose) polymerase (PARP)inhibitor

New

Pro

du

ct H

igh

ligh

ts

NH

S

O

PNU-37883A (U-37883A): Selective vascularATP-sensitive K+ Kir6.1/SUR2B channel blocker

Prod. Code P 0248Selective blocker (IC50 = 6 µM) against HEK-293 cells stably expressing putative smoothmuscle Kir6.1/SUR2B channels [1]; IC50 = 5 µM against Kir6.1/Kir6.2 chimeric channelsco-expressed with SUR1 in Xenopus oocytes[2]. PNU-37883A is more potent than thesulfonylurea drug Glybenclamide (Glyburide)

at inhibiting Kir6.1/SUR2B channels expressed in the brain (50-fold, as measured by half-maximal concentration to achievechannel inhibition), kidney (9-fold, as measured by increased uri-nary sodium excretion), and vascular smooth muscle (6-fold, asmeasured by a decrease in blood pressure, induced by an ATP-sensitive K+ channel opener, Pinacidil) [3,4].

References1. Cui, Y., et al., Different molecular sites of action for the KATP channel

inhibitors, PNU-99963 and PNU-37883A. Br. J. Pharmacol., 139, 122-128(2003).

2. Kovalev, H., et al., Molecular analysis of the subtype-selective inhibition ofcloned KATP channel by PNU-37883A. Br. J. Pharmacol., 141, 867-873(2004).

3. Ludens, J.H., et al., Renal and vascular effects on chemically distinct ATP-sensitive K+ channel blockers in rats. J. Cardiovasc. Pharmacol., 25, 404-409(1995).

4. Lin, Y-J., et al., U-37883A potently inhibits dopamine-modulated K+

channels on rat striatal neurons. Eur. J. Pharmacol., 352, 335-341 (1998).

Related Products

Product Name Descriptor Prod. Code

Glybenclamide Selective ATP-sensitive K+ channel blocker G 0639

Pinacidil K+ channel activator P-154

HCl

NN

NH

O

Available First from Sigma-RBI!

Psora-4: Potent KV1.3 K+ channel blocker

Prod. Code P 9872Psora-4 is the most potent blocker of KV1.3 K+ channels described to date. Itblocks these channels in a use-dependentmanner displaying an EC50 value of 3 nM,and exhibits 17- to 70-fold selectivity forKV1.3 over KV1.1, KV1.2, KV1.4 and KV1.7.It shows no effect on the ether-a-go-go-related channel, KV3.1, the Ca2+-activatedK+ channels or neuronal Nav1.2 channels. It

has been shown to suppress proliferation of human and ratmyelin-specific effector memory T-cells displaying EC50 values of25 nM and 60 nM, respectively.

References1. Vennekamp, J., et al., Kv1.3-blocking 5-phenylalkoxypsoralens: a new class

of immunomodulators. Mol. Pharmacol., 65, 1364-1374 (2004).2. Chandy, K.G., et al., K+ channels as targets for specific immunomodula-

tion. Trends Pharmacol. Sci., 25, 280-289 (2004).

Additional K+ Channel Blockers

Product Name Prod. Code

Agitoxin-1, recombinant, expressed in E. coli A 5229

Margatoxin from Centruoides margaritatus (scorpion) M 8278

Margatoxin from Centruoides margaritatus (scorpion) M 8437recombinant, expressed in E. coli

O O O

O

Available First from Sigma-RBI!

NNC 55-0396: Selective T-type Ca2+ channelblocker

Prod. Code N 0287Nonhydrolyzable analog ofmibefradil; IC50 in HEK293cells is 7 µM. Does notaffect the voltage-depend-ent activation of T-type Ca2+

currents, but changes theslope of the steady-stateinactivation curve.

Reference1. Huang, L., et al., NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-

yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2,3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride]: a new selective inhibitorof T-type calcium channels. J. Pharmacol. Exp. Ther., 309, 193-199 (2004).

Additional T-type Ca2+ Channel Blockers

Product Name Prod. Code

Mibefradil dihydrochloride M 5441

Penfluridol P 3371

Kurtoxin K 1514

F

H3C CH3

O

N

CH3 N

HN

2 HCl

O

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

20N

ew P

rod

uct

Hig

hlig

hts

Diethylumbelliferyl phosphate (UBP;DEUP): Cholesteryl ester hydrolase inhibitor

Prod. Code D 7692

An effective inhibitor of the cholesterylester hydrolase in intact MA-10 Leydigtumor cells (IC50 = 11.6 µM in(Bu)2cAMP-stimulated, cholesterylester-loaded MA-10 cells) [1]. Blockssteroidogenesis mainly by preventing

cholesterol transport into the mitochondria of steroidogenic cells[1]. In Fu5AH cells, UBP caused a 72% decrease in the cellularfree cholesterol/cholesteryl ester and inhibition of triglycerides(TG), but did not interfere with esterified cholesterol and TG syn-thesis nor did it cause cellular toxicity in doses up to 120 µg/ml[2,3]. Inhibitors of cholesterol esterase are anticipated to limitthe absorption of dietary cholesterol [4].

References1. Gocze, P.M., et al., A cholesteryl ester hydrolase inhibitor blocks choles-

terol translocation into the mitochondria of MA-10 Leydig tumor cells.Endocrinology, 131, 2972-2978 (1992).

2. Delamatre, J.G., et al., Evidence that a neutral cholesteryl ester hydrolaseis responsible for the extralysosomal hydrolysis of high-density lipoproteincholesteryl ester in rat hepatoma cells (Fu5AH). J. Cell Physiol., 157, 164-168 (1993).

3. Kellner-Weibel, G., et al., Evidence that newly synthesized esterified cho-lesterol is deposited in existing cytoplasmic lipid inclusions. J. Lipid Res.,42, 768-777 (2001).

4. Heidrich, J.E., et al., Inhibition of pancreatic cholesterol esterase reducescholesterol absorption in the hamster. BMC Pharmacol., 4, 5 (2004).

OOP

O

C2H5O

C2H5O

O

CH3

MEDICA 16: ATP-citrate lyase inhibitor; potenttriacylglycerol-lowering agent

Prod. Code M 5693ATP-citrate lyase is the main enzymeresponsible for supplying acetyl-CoA tomany tissues, and most notably in adi-pose tissue and liver where de novo syn-thesis of fatty acids is very active, espe-cially when glucose is in excess [1].Because ATP-citrate lyase is involved in

both the fatty acid and cholesterol synthesis pathways, it hasbeen suggested that inhibition of this enzyme may be a drugtarget for hyperlipidemia [1]. One compound that supports thissuggestion is MEDICA 16. When MEDICA 16 was given toJCR:LA-cp rats (0.25% (wt/wt)) starting at weaning up to threeweeks of age, (JCR:LA-cp rats develop extreme obese/insulin-resistant syndrome by 12 weeks of age), their plasma lipidsdecreased dramatically and their food intake and body weightreturned to normal levels by 8 weeks of age [2]. In addition,insulin levels were significantly decreased, and plasma triacyl-glycerol concentrations were maintained at the same level as thecontrol lean rats [2].

References1. Groot, P.H.E., et al., ATP-citrate lyase: A potential target for hypolipidemic

intervention. Curr. Med. Chem. Immunol. Endocr. Metab. Agents, 3, 211-217 (2003).

2. Russell, J.C., et al., Development of insulin resistance in the JCR:LA-cp rat:role of triacylglycerols and effects of MEDICA 16. Diabetes, 47, 770-778(1998).

COOH

COOH

H3C CH3

H3C CH3

S-15176 difumarate salt: Carnitinepalmitoyltransferase (CPT-1) inhibitor; antioxidantand anti-ischemic agent

Prod. Code S 5944

IC50 for carnitine palmitoyl-transferase (CPT-1) in hearthomogenate is 16.8 µM.Inhibits in vitro lipid peroxi-dation (0.3 µM) in liverfrom animals subjected to2 hr of liver injury inducedby warm ischemia-reper-fusion. The shift from fattyacid to glucose oxidation

may contribute to anti-ischemic effect. Also used to inhibit mito-chondrial permeability transition and to prevent onset of apopto-sis by preventing collapse of the electrochemical gradient acrossthe mitochondrial membrane.

References1. Hamdan, M., et al., Inhibition of mitochondrial carnitine palmitoyltrans-

ferase-1 by a trimetazidine derivative, S-15176. Pharmacol. Res., 44, 99-104(2001).

2. Rupp, H., et al., The use of partial fatty acid oxidation inhibitors for meta-bolic therapy of angina pectoris and heart failure. Herz, 27, 621-636 (2002).

3. Settaf, A, et al., S-15176 reduces the hepatic injury in rats subjected toexperimental ischemia and reperfusion. Eur. J. Pharmacol., 406, 281-292(2000).

4. Eliadi, A., et al., S-15176 inhibits mitochondrial permeability transition viaa mechanism independent of its antioxidant properties. Eur. J. Pharmacol.,468, 93-101 (2003).

CH3O

CH3O

NN

S

OH

CH3O

2C4H4O4

(CH3)3C

C(CH3)3

A-350619 hydrochloride: Novel, solubleguanylyl cyclase activator

OHN

NCH3

CH3

S

Cl

HCl

Prod. Code A 6604The soluble guanylyl cyclase (sGC)receptor is a major receptor for nitricoxide (NO). Guanylyl cyclase convertsGTP to cyclic GMP affecting such physi-ological processes as smooth musclerelaxation, neurotransmission, inhibitionof platelet aggregation and immuneresponse. A-350619, an activator of

sGC, modulates the catalytic properties of sGC [increases Vmaxfrom 0.1 to 14.5 µmol/min/mg (145-fold increase), lowers Kmfrom 300 to 50 µM (6-fold decrease)]. A-350619 has also beenshown to relax rabbit corpus cavernosum tissue strips in a dose-dependent manner with EC50 of 80 µM (vs 50 µM for anothersGC inhbitor, YC-1). Moreover, A-350619 has been shown toinduce penile erection in a conscious rat model (1 µmol/kg)suggesting that activation of sGC could be used as an alternatemethod of enhancing the effect of NO for the treatment ofsexual dysfunction.

Sold under license from Abbott Laboratories

Reference1. Miller, L.N., et al., A-350619: a novel activator of soluble guanylyl cyclase.

Life Sci., 72, 1015-1025 (2003).

Related Products

Product Name Descriptor Prod. Code

Isoliquiritigenin Guanylyl cyclase activator I 3766

NS 2028 Guanylyl cyclase inhibitor N-211

ODQ Guanylyl cyclase inhibitor O 3636

YC-1 Guanylyl cyclase activator Y-102

OHN

NCH3

CH3

S

Cl

HCl

21

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

New

Pro

du

ct H

igh

ligh

ts

NU7026: Potent, selective, cell-permeable DNA-PK(DNA-dependent protein kinase) inhibitor

Prod. Code N 1537

ATP-competitive inhibitor of DNA-PK. IC50of 230 nM against DNA-PK purified fromHela cell nuclear extract vs. IC50 of 13 µMand >100 µM for recombinant PI3K (phos-phoinositide 3-kinase) and ATM (ataxiatelangiectasia-mutated) from HeLa cellnuclear extract, respectively.

References1. Hollick, J.J., et al., 2,6-disubstituted pyran-4-one and thiopyran-4-one

inhibitors of DNA-Dependent protein kinase (DNA-PK). Bioorg. Med.Chem. Lett., 15, 3083-3086 (2003).

2. Willmore, E., et al., A novel DNA-dependent protein kinase inhibitor,NU7026, potentiates the cytotoxicity of topoisomerase II poisons used inthe treatment of leukemia. Blood, 103, 4659-4665 (2004).

3. Veuger, S.J., et al., Radiosensitization and DNA repair inhibition by thecombined use of novel inhibitors of DNA-dependent protein kinase andpoly(ADP-ribose) polymerase-1. Cancer Res., 15, 6008-6015 (2003).

Additional DNA-PK Inhibitor

Product Name Prod. Code

4,5-Dimethoxy-6-nitrobenzaldehyde D 9192

O

O

NO

TG003: Potent, selective, reversible and ATP-competitive Cdc2-like kinase (Clk) inhibitor

Prod. Code T 5575Displays an IC50 of 200 nM and anIC50 of between 15-20 nM forrecombinant Clk2 and Clk1/Sty andClk4, respectively. Inhibits SF2/ASF-dependent splicing of β-globin pre-mRNA in vitro by suppression of Clk-mediated phosphorylation. TG003also suppresses serine/arginine-rich

protein phosphorylation, dissociation of nuclear speckles, andClk1/Sty-dependent alternative splicing in mammalian cells.TG003 acts on recombinant Clk1/Sty competitively with ATP(Km = 3.35 µM) with a Ki value of 10 nM. Administration ofTG003 rescued the embryonic defects induced by excessive Clkactivity in Xenopus. Thus, TG003 may be applicable for thetherapeutic manipulation of abnormal splicing.

Reference1. Muraki, M., et al., Manipulation of alternative splicing by a newly devel-

oped inhibitor of clks. J. Biol. Chem., 279, 24246-24254 (2004).

N

S

H3CO

CH3

O

CH3

L-NASPA: Competitive mammalian lyso-phosphatidic acid (LPA) receptor agonist

Prod. Code P 0247LPA mimetic; potent reversible antago-nist of LPA receptors expressed inXenopus oocytes [1]. Used to character-ize the LPA receptor (Edg-7). In humanosteosarcoma MG63 cells, L-NASPA(0.1-10 µM) induced a rapid and tran-sient increase in intracellular Ca2+ in aconcentration-dependent manner (EC50

of 0.5 µM) [2]. In human breast carcinoma MDA MB231 cells, L-NASPA, like LPA, mobilized Ca2+ and inhibits activation ofadenylyl cyclase, acting as a high potency LPA mimetic [3].

References1. Bittman, R., et al., Inhibitors of lipid phosphatidate receptors: N-palmitoyl-

serine and N-palmitoyl-tyrosine phosphoric acids. J. Lipid Res., 37, 391-398(1996).

2. Jan, C.R., et al., Novel effect of N-palmitoyl-L-serine phosphoric acid oncytosolic Ca2+ levels in human osteoblasts. Pharmacol. Toxicol., 93, 71-76(2003).

3. Hooks, S.B., et al., Characterization of a receptor subtype-selectivelysophosphatidic acid mimetic. Mol. Pharmacol., 53, 188-194 (1998).

Related Product

Product Name Description Prod. Code

Lysophosphatidic acid (LPA) Endogenous LPA1 and LPA2 L 7260receptor agonist

NH

O

O

OHOP

HO

OHO

CH3

Tyrosine Kinase Assay Kit

Prod. Code CS0730

Protein tyrosine kinases (PTKs) comprise a large and diverse multi-gene family. The functions of these enzymes involve regulation ofcell to cell signaling associated with growth, differentiation, adhe-sion, motility and death. Tyrosine kinases play significant roles inthe development of many disease states, including diabetes andcancer.

The Tyrosine Kinase Assay Kit assay is based on immunoprecipita-tion of the tyrosine kinase of interest using a specific anti-tyrosinekinase protein antibody. As the kit was designed for use with anytyrosine kinase, researchers should use an immunoprecipitationantibody corresponding to the protein of interest. The immuno-precipitated kinase is incubated with its substrate (poly [Glu,Tyr]4:1) in the presence of [γ32P]-ATP and the radioactive-phosphateincorporation is measured.

The kit also offers a short and easy alternative detection methodwith no requirement for a radioactively labeled ATP or secondaryantibody. Suitable for dot blot analysis.

Size: Sufficient for 50 reactions

Components:• Monoclonal Anti-Phosphotyrosine-Peroxidase antibody

produced in mouse, 1 vial• Wash buffer 10X, 8 ml• Assay buffer for tyrosine kinase activity, 1 mL • EZviewTM Red Protein A affinity gel, 1 mL • Epidermal growth factor receptor, 500 units • P81 cellulose phosphate squares, 10 each

22

®

®

Celltr

ansm

issi

ons

Vol 2

1, N

o. 1

, 20

05

Ord

er:

1-80

0-32

5-30

10

Tech

nica

l Ser

vice

: 1-

800-

325-

5832

s

igm

a-al

dric

h.co

m/c

ells

igna

ling

New

Pro

du

ct H

igh

ligh

tsMonoclonal Anti-Importin α1

Prod. Code: I 9658Clone Name: Clone 1A6, developed in rat Product Form: Purified rat immunoglobulinImmunogen: Recombinant mouse importin α1Isotype: IgG2aSpecies Cross Reactivity: human, mouse, canine, chicken

Importin α is a nuclear transport adaptor protein and is classifiedinto three subfamilies: the SRP1-like subfamily (containing theSRP1; Importin α5, α6, and α7), the Rch1-like subfamily (con-taining Rch1, Pendulin, Importin α1, and α2); and Importinα3/Qip1-like subfamily (containing Importin α3 and α4).Importin α links the import receptor, importin β, with cargo pro-teins containing a classical nuclear localization signal (NLS).Formation of the Importin β/Importin α/cargo complex triggersthe binding of importin β to the nuclear pore complex (NPC) andthe subsequent import of the entire complex into the nucleus.Inside the nucleus, the cargo protein and importin α are releasedfrom the complex upon binding of Ran-GTP to importin β.Importin α is recycled back to the cytoplasm by CAS (cellularapoptosis susceptibility) protein, an importin α specific exportreceptor.

Applications: Immunoblotting (~60 kDa) and immunocyto-chemistry

Reference1. Sakaguchi, N., et al., Generation of a rat monoclonal antibody specific for

importin α3/Qip1. Hybrid Hybridomics, 22, 397-400 (2003).

Related Antibodies

Product Name Clone Host Prod. Code

Monoclonal Anti-Importin, α3 3D10 Rat I 9783

Monoclonal Anti-Importin, α5/7 2D9 Rat I 9908

Related Proteins

Product Name Prod. Code

Importin α2, human, recombinant I 9656

Importin β1, human, recombinant I 9781

Importin

1 2 3

M.W.

75 kDa_

50 kDa_

25 kDa_

α1 α5/7 α3

Detection of Importin α1, α3 and α5/7 inHeLa cells.Total cell extracts from HeLa cells were testedby immunoblotting using three importin-specificrat monoclonals. Lane 1: Anti-Importin α1 (Prod. Code I 9658)Lane 2: Anti-Importin α5/7 (Prod. Code I 9908)Lane 3: Anti-Importin α3 (Prod. Code I 9783)

Monoclonal Anti-PINCH1

Prod. Code: P 8996Clone Name: PINCH-C58, developed in mouseProduct Form: Purified mouse immunoglobulinImmunogen: Synthetic peptide corresponding to amino acids

281-299 of human PINCH-1Isotype: IgG1Species Cross Reactivity: human, mouse, rat, hamster,

monkey, bovine and canine

Cell adhesion to extracellular matrix (ECM) is an importantprocess that controls cell morphology, proliferation, migration,differentiation and survival. Transduction of extracellular matrixsignals through integrins influences intracellular and extracellularfunctions, and requires interaction of integrin cytoplasmicdomains with cellular proteins. ILK (integrin-linked kinase) inter-acts with the cytoplasmic domain of β1- and β3-integrins and isimplicated in integrin, growth factor and Wnt signaling path-ways [1-3]. ILK has three structurally conserved domains; the C-terminal domain contains a protein kinase catalytic site and abinding site for the integrin β1 subunit; the PH-like domain ofILK binds PtdIns(3,4,5)P3 and participates in the regulation of thekinase activity [4]; the N-terminal domain contains four ANKrepeats responsible for interaction with the LIM adaptor proteinPINCH (particularly interesting new Cys-His protein; a.k.a. LIMand senescent cell antigen-like domains) [5,6]. The interactionwith PINCH proteins (PINCH-1 and 2) is important for localiza-tion of the ILK protein to cell matrix contact sites.

Applications: Immunoblotting (~37 kDa), immunoprecipitationand ELISA

References1. Hannigan, G.E., et al., Regulation of cell adhesion and anchorage-depend-

ent growth by a new β1-integrin-linked protein kinase. Nature, 379, 91-96(1996).

2. Li, F., et al., Identification and characterization of a mouse protein kinasethat is highly homologous to human integrin-linked kinase. Biochim.Biophys. Acta, 1358, 215-220 (1997).

3. Wu, C. and Dedhar, S., Integrin-linked kinase (ILK) and its interactors: anew paradigm for the coupling of extracellular matrix to actin cyto-skeleton and signaling complexes. J. Cell. Biol., 155, 505-510 (2001).

4. Delcommenne, M., et al., Phosphoinositide-3-OH kinase-dependent regu-lation of glycogen synthase kinase 3 and protein kinase B/AKT by the inte-grin-linked kinase. Proc. Natl. Acad. Sci. USA, 95, 11211-11216 (1998).

5. Tu, Y., et al., The LIM-only protein PINCH directly interacts with integrin-linked kinase and is recruited to integrin-rich sites in spreading cells. Mol.Cell. Biol., 19, 2425-2434 (1999).

6. Wu, C., Integrin-linked kinase and PINCH: partners in regulation of cell-extracellular matrix interaction and signal transduction. J. Cell. Sci., 112,4485-4489 (1999).

Related Antibodies

Product Name Clone Host Prod. Code

Monoclonal Anti-Pinch-1 PINCH-N173 Mouse P 9371

Monoclonal Anti-ILK 65 Mouse I 0783

Anti-ILK -- Rabbit I 1907

Antibody Product Guide• Over 3,000 quality antibodies

all rigorously tested• Hundreds of NEW antibodies• Organized by area of study

for easy reference• World-renowned technical

service• Timely delivery anywhere

Ion Channel Brochure• Brochure covering 17 ion

channel-related topics such as:AquaporinsCyclic Nucleotide-GatedChannelsIon Exchangers and Co-TransportersLigand-Gated ChannelsVoltage-Gated Channels

• Over 600 products, including over 100 NEW products

Alzheimer’s Poster detailing:• α-Secretase pathway• β-Secretase pathway• Immune response• Over 200 products listed

New Literature Available from Sigma-RBI

Fill out & return the Business Reply Card to receive your new Sigma-RBI Literatureor visit us online at sigma-aldrich.com/cellsignaling_os2

(007)

(HEC)

(HJI)

2323

L E A D E R S H I P I N L I F E S C I E N C E , H I G H T E C H N O L O G Y A N D S E R V I C ESIGMA-ALDRICH CORPORATION • BOX 14508 • ST. LOUIS • MISSOURI 63178 • USAsigma-genosys.com

PEPscreen® is a registered trademark of Sigma-Genosys, L.P.

Get Results FAST...PEPscreen®: Custom Peptide Libraries

Sigma-Genosys has developed a proprietary peptide synthesis platform using state-of-the-arttechnology. This technology allows the fastest and most efficient high-throughput parallelsynthesis of milligram quantities of peptide libraries. The PEPscreen® service provides researcherswith the ability to order large numbers of extremely affordable custom peptides delivered inless than 7 business days.

Ordering Information

For more information, please refer to www.sigma-genosys.com/CT305

For technical inquiries, please contact peptides@sial.com

Place your order by emailing your sequences in an 8-channel orientation (peptide #1 = A1, peptide #2 = B1, peptide #8 = H1, peptide #9 = A2, etc.)

Product Specifications

• Delivery: 7 business days or less

• Quantity: 0.5–2 mg

• Length: 6–20 amino acids

• QC: MALDI-TOF MS on 100% of peptides

• Format: lyophilized in 96-well tube racks

• Shipped with electronic PDF files of QC data and peptide sequence directory

• Minimum order size is 48 peptides

Features and Benefits

• Compatible with robotic automation for resuspension anddispensing in screening applications

• Ideal for performing solution-phase assays

• Suitable for robotic production of peptide microarrays

• N-Terminal modifications and nonstandard residue availabilityallows for maximum flexibility in project design

• Delivery of product in less than 7 days allows for faster target identification

Modifications Available

• N-Terminal acetylation

• N-Terminal biotin

• N-Terminal fluorescein

• D-Amino acids

• Nonstandard residues

• Phosphorylated residues

• C-Terminal acid or amide

Editorial BoardScientific Editor: Keith J. Watling, Ph.D.Managing Editor: Mona M. Scrofano, Ph.D.Layout/Design/Production: Diane Ward

Three Strathmore Road, Natick, MA 01760-2447 USANet: sigma-aldrich.com© 2005 by Sigma-RBI, A Member of the Sigma-Aldrich® Family. All rights reserved. No part of this publication may bereproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, photocopying, record-ing, or otherwise, without the prior written permission of the copyright holder. Sigma brand products are sold throughSigma-Aldrich, Inc. Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this andother Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use.Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip.

The SIGMA-ALDRICH Family

World Headquarters • 3050 Spruce St., St. Louis, MO 63103 • (314) 771-5765

Order/Customer Service 1-800-325-3010 • Fax 1-800-325-5052Technical Service 1-800-325-5832 • sigma-aldrich.com/techservice

Development/Bulk Manufacturing Inquiries 1-800-336-9719

HXP02001-502641

0035

We are committed to the success of our Customers, Employees and Shareholders through leadership in Life Science, High Technology and Service.

©2005 Sigma-Aldrich Co. Printed in USA Sigma brand products are sold through Sigma-Aldrich, Inc.Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of the invoice or packing slip. SIGMA and - are registered trademarks of Sigma-Aldrich Co. and its division Sigma-Aldrich Biotechnology LP. Riedel-de Haën®: trademark under license from Riedel-de Haën GmbH.

ArgentinaSIGMA-ALDRICH DE ARGENTINA, S.A.Tel: 54 11 4556 1472Fax: 54 11 4552 1698

AustraliaSIGMA-ALDRICH PTY., LIMITEDFree Tel: 1800 800 097 Free Fax: 1800 800 096Tel: 612 9841 0555Fax: 612 9841 0500

AustriaSIGMA-ALDRICH HANDELS GmbHTel: 43 1 605 81 10Fax: 43 1 605 81 20

BelgiumSIGMA-ALDRICH NV/SA.Free Tel: 0800-14747Free Fax: 0800-14745Tel: 03 899 13 01Fax: 03 899 13 11

BrazilSIGMA-ALDRICH BRASIL LTDA.Tel: 55 11 3732-3100Fax: 55 11 3733-5151

CanadaSIGMA-ALDRICH CANADA LTD.Free Tel: 800-565-1400Free Fax: 800-265-3858Tel: 905-829-9500Fax: 905-829-9292

ChinaSIGMA-ALDRICH CHINA INC.Tel: 86-21-6386 2766Fax: 86-21-6386 3966

Czech RepublicSIGMA-ALDRICH s.r.o.Tel: 246 003 200Fax: 246 003 291

DenmarkSIGMA-ALDRICH DENMARK A/STel: 43 56 59 10Fax: 43 56 59 05

FinlandSIGMA-ALDRICH FINLANDTel: 09-3509250Fax: 09-350-92555

FranceSIGMA-ALDRICH CHIMIE S.à.r.l.Tel appel gratuit: 0800 211 408Fax appel gratuit: 0800 031 052

GermanySIGMA-ALDRICH CHEMIE GmbHFree Tel: 0800-51 55 000Free Fax: 0800-649 00 00

GreeceSIGMA-ALDRICH (O.M.) LTDTel: 30 210 9948010Fax: 30 210 9943831

HungarySIGMA-ALDRICH KftTel: 06-1-235-9054Fax: 06-1-269-6470Ingyenes zöld telefon: 06-80-355-355Ingyenes zöld fax: 06-80-344-344

IndiaSIGMA-ALDRICH CHEMICALS PRIVATE LIMITEDTelephoneBangalore: 91-80-5112-7272New Delhi: 91-11-2616 5477Mumbai: 91-22-2570 2364Hyderabad: 91-40-5531 5488FaxBangalore: 91-80-5112-7473New Delhi: 91-11-2616 5611Mumbai: 91-22-2579 7589Hyderabad: 91-40-5531 5466

IrelandSIGMA-ALDRICH IRELAND LTD.Free Tel: 1800 200 888Free Fax: 1800 600 222Tel: 353 1 4041900Fax: 353 1 4041910

IsraelSIGMA-ALDRICH ISRAEL LTD.Free Tel: 1-800-70-2222Tel: 08-948-4100Fax: 08-948-4200

ItalySIGMA-ALDRICH S.r.l.Telefono: 02 33417310Fax: 02 38010737Numero Verde: 800-827018

JapanSIGMA-ALDRICH JAPAN K.K.Tokyo Tel: 03 5796 7300Tokyo Fax: 03 5796 7315

KoreaSIGMA-ALDRICH KOREATel: 031-329-9000Fax: 031-329-9090

MalaysiaSIGMA-ALDRICH (M) SDN. BHDTel: 603-56353321Fax: 603-56354116

MexicoSIGMA-ALDRICH QUÍMICA, S.A. de C.V.Free Tel: 01-800-007-5300Free Fax: 01-800-712-9920

The NetherlandsSIGMA-ALDRICH CHEMIE BVTel Gratis: 0800-0229088Fax Gratis: 0800-0229089Tel: 078-6205411Fax: 078-6205421

New ZealandSIGMA-ALDRICH PTY., LIMITEDFree Tel: 0800 936 666Free Fax: 0800 937 777

NorwaySIGMA-ALDRICH NORWAY ASTel: 23 17 60 60Fax: 23 17 60 50

PolandSIGMA-ALDRICH Sp. z o.o.Tel: (+61) 829 01 00Fax: (+61) 829 01 20

PortugalSIGMA-ALDRICH QUÍMICA, S.A.Free Tel: 800 202180Free Fax: 800 202178Tel: 21 9242555Fax: 21 9242610

RussiaSIGMA-ALDRICH RUSSIAOOO SAF-LABTel: 095 975-1917/3321Fax: 095 975-4792

SingaporeSIGMA-ALDRICH PTE. LTD.Tel: 65-67791200Fax: 65-67791822

South AfricaSIGMA-ALDRICH SOUTH AFRICA (PTY) LTD.Free Tel: 0800 1100 75Free Fax: 0800 1100 79Tel: 27 11 979 1188Fax: 27 11 979 1119

SpainSIGMA-ALDRICH QUÍMICA S.A.Free Tel: 900 101376Free Fax: 900 102028Tel: 91 661 99 77Fax: 91 661 96 42

SwedenSIGMA-ALDRICH SWEDEN ABTel: 020-350510Fax: 020-352522Outside Sweden Tel: +46 8 7424200Outside Sweden Fax: +46 8 7424243

SwitzerlandFLUKA CHEMIE GmbHSwiss Free Call: 0800 80 00 80Tel: +41 81 755 2828Fax: +41 81 755 2815

United KingdomSIGMA-ALDRICH COMPANY LTD.Free Tel: 0800 717181Free Fax: 0800 378785Tel: 01747 833000Fax: 01747 833313

United StatesSIGMA-ALDRICHP.O. Box 14508St. Louis, Missouri 63178Toll-free: 800-325-3010Call Collect: 314-771-5750Toll-Free Fax: 800-325-5052Tel: 314-771-5765Fax: 314-771-5757Internet:sigma-aldrich.com

sigma-aldrich.com

Avastin® is a registered trademark of Genentech, Inc.Macugen® is a registered trademark of Eyetech Pharmaceuticals, Inc.Angiozyme® is a registered trademark of Ribozyme Pharmaceuticals Incorporated