Embed Size (px)
Citation preview
G-Protein-coupledReceptor
PKA
CBP CCCBBBBPPPP CREB P
RERE
ATP cAMP
AC
Gα
Desensitiza-tion
Ca2+
PKC
NFAT P
NFAT
P
Calcineurin
MAPK/ERK 1/2 P
2 PP
MEK 1/2 P
2 PP
Raf
DAG
IP3
PIP2
PD 98059UO126 1
3
mPKCI 7
IKKγIKKα
IKKγIKKβ
p65 p50 Ub
P
p6555 p5500UUb UUb
PIκB
Ub
P
UUb UUb
PIκB
Proteasome- Degradation
PLC
αP
P α P
Ligand
GRK PP
GRK
G /γ
P
P P
RERE
P
P
p65
P
5 p50
Des
GRK
-Arrestin
P P P
Ligand
Raf ERK RK
MEK
P PP PP
RRaaff ER
MMMEK -Arrestin
Receptor-Internalization
Signaling
EGF Receptor
EGF EGF
P P P
Shc
MMP
JAK1 JAK1 JAK2
Shc
GRB2 GRB22
SOS
Src
MMP
Hb EGF
ELK-1 P
SRE
P
P
STAT PPPPPP
P STAT
P
P
STAT PPPP P
PP STAT
GAS, ISRE
MAPK/ERK 1/2 P 2 P P
MEK 1/2 P
2 P P
Raf
Ras Rac
SAPK/JNK P P P
SEK 1/MKK 4 4 P
MEKK 1/4
C-Jun P PPP P
AP-1
Cytokine Receptor
JAK1 JAK2
Cytokine
STAT
STAT
P PP
P P P
PP
JAK1 P
PP
JAK2 P
Cytokine
P
P
STAT PPPP
P STAT
P
P
STAT PPPPPP
P STAT
GAS, ISRE
TGF Receptor
Type II Type I pe II TypTy
TGF
P
I pe Ip TGF
SARASARA
SMAD 2/3
SMAD 2/33 P
SMAD 7
SMAD 6
SMURF
SMAD 4S
SMAD 2/3 P
SMAADD 44SMAAADDD 444
SMMAAADDD 222///33333SMAD 2/3
PP 3 P
SMAD 4
SMAD 4
SMAD 2/3 P
SMAADDD 444MAAADDD 44
MMAADDD 222///333333 PSMAD 2/3
PP 3 P
SMM
Transcriptionfactors
SSSMSM
SS
Transcriptionfactors
Co-Factors
SBE
Fas/CD95Receptor
Receptor-Internalization
ceptor RecReRnternalizatioIIntertent
FasL
FADDFADFADFADDDD
Caspase-8/-10
non
FasL
FADDFADD
Caspase-8/-10
FasL FasL
FasL
FADDFADD
Caspase-8/-10
FADDFADD
Caspase-8/-10
FasL
Latrunculin A
0
Ub Ubb FLIP
Proteasome-Degradation
Caspase-3
ICAD ICAD CAD
CAD AIF
AIF
PARP
DNAFragmentation
DNARepair
Bid
Bax
Bcl-2
x
tBid
Caspase-7
Apaf-1 Apaf-1
Caspase-9
Apoptosis
CytC
CIAP Smac/DIABLO
TRAILReceptor
TRAIL
FADDFADD
Caspase-8/-10
TNF-Receptor 1
TNFα
TRADD RIP
TRAF 2 TRADD
FADD RIP TRAF 2 Caspase-8/-10
Complex II
Complex I
Modification
TRAF
n
ASK-1
MKK 3/6 P
p38 P
88 P
SRE
P PP Elk-1
MKK 4/7 P
JNKP
P
Itch P
TNF-Receptor 2
MEKK 3
NIK
TRAF 2
TNFα
IKKγIKKα
IKKγIKKβ
p65 p50 Ub
P
p6555 p5500 UUb UUb
P IκB
Ub
P
UUb UUb
PIκB
Proteasome-Degradation
IKKα IKKα
Ub RelB UUbB p100 bUUb00 P
RelB B p52
RE
RelB B p52
13, 15
1
12
16
13–15
1
14
1
1
5
7
12
7
1
12
6, 9
8
6, 9
2, 3
7
4, 17–23
17–23
17–23
17–23
17–23
17–23
17–23
17–23
17–23
10, 11
PKI2
PD 98059UO126 1
3
Z-VAD-FMKZ-IETD
5
Z-VAD-FMKZ-IETD
5
SB 2035804
Ac-DEVD-CHO6
Z-VAD-FMK5
Z-VAD-FMKZ-IETD
5
2–4, 6
2–4, 6 1, 3
7
7
11
8
3, 5
9
1, 3
10
Cell Signaling Pathway Analysis using Bioluminescence
Promega
Signal transduction is one of the most widely studied areas
in molecular biology. Extracellular information is translated
into an intracellular response, often through elaborate net-
works of interwoven signaling cascades. Gaining a better
understanding of these signaling pathways may lead to
development of new therapeutics. This brochure is designed
to help you choose the right Promega assays to monitor
your signaling pathway of interest.
There are several methods for monitoring receptor-mediat-
ed (e.g., G-protein-coupled receptors (GPCR)) activation of
cellular signaling, including those that use response ele-
ments upstream of a primary reporter to measure transcrip-
tional activation. Genetic reporter assays allow you to
detect modulation of gene expression by a regulatory
sequence of interest. For example, reporter assays can be
used to monitor the effect of a particular treatment on a
specific promoter or other regulatory sequence. Additionally,
libraries of regulatory sequences can be screened using
genet ic reporters to allow you to observe constellations of
regulatory events that result from a treatment strategy or
test compound.
A well known example for cell signaling occurs via GPCR.
The receptors represent one of the most important classes
of drug targets. Major G-protein families signal via second
messengers like cAMP, which in turn activate a range of
effector systems to change cell behavior and gene transcrip-
tion. Transcriptional activation can be monitored using
reporter assays.
Promega offers a range of range of ready-to-use vectors and
cell lines designed to measure cell signaling events. Features
include:
• A Choice of Reporter Genes: Firefly (luc2), Renilla (hRluc)
and NanoLuc® (Nluc) Luciferase
• Pre-designed Vectors: No need to clone and validate an
assay.
• Increased Reporter Gene Expression: Luciferase genes
are codon optimized for enhanced expression in mam-
malian cells.
• Reduced Background and Risk of Expression Artifacts:
Cryptic DNA regulatory elements and transcription fac-
tor binding sites are removed.
• Improved Temporal Response: Rapid Response™ techno-
logy using destabilized luciferase genes.
• Easy Transition from Transient to Stable Cells: Choice of
mammalian-selectable markers.
2
Cell Signaling Pathway Analysis using Bioluminescence
Promega offers various ready-to-use firefly luciferase reportervectors based on the pGL4 Vector series. These vectors all con-tain the destabilized firefly luciferase luc2P gene, a minimalpromoter (minP), and a hygromycin resistant cassette (Hyg)selectable marker. The “P” in Luc2P indicates the addition ofdestabilizing sequences to influence the cellular half-life of theLuc2 protein. The shorter half-life means that the reporteractivity follows the transcriptional activity more closely than itwould with a more stable protein. The various firefly luciferasereporters are distinguishable by the Response Element (RE)present upstream of the minimal promoter controllingluciferase expression. Activation of the RE leads to proportionalexpression of the firefly luciferase gene, which can be meas-ured using luciferase assay reagents.
For more information on the luciferase assay reagents availablefrom Promega, please visit: www.promega.com/reporter-assays.For detailed information on the pGL4 Vectors, please visit:www.promega.com/pgl4-vectors.
Applications of Luciferase Reporter Assays• Cell signaling.• Promoter analysis • Transcription regulation.• Virus-cell interactions.• Compound screening.• Post-translational modifications.• GPCR signaling.
I . Firefly Luciferase Pathway Reporter Vectors and Stable Cell Lines
3
GloResponse™ Cell Lines
cAMP/PKA
Calcium/Calcineurin
NF-kB
Activator/PathwayCREB
NFAT
NF-kB
Transcription FactorE8500
E8510
E8520
Catalog #GloResponse™ CRE-luc2P HEK293 Cell LineGloResponse™ NFAT-RE-luc2PHEK293 Cell LineGloResponse™ NF-kB-RE-luc2PHEK293 Cell Line
Cell Line Cyclic AMP Response Element
Nuclear Factor of Activated T-Cells(NFAT) Response ElementNuclear Factor kB Response Element
Response Element
Calcium/Calcineurin
cAMP/PKADNA DamageEndoplasmic Reticulum StressHeat ShockHeavy Metal StressHypoxiaIL3IL6INF-α
MAP/ERKMAPK/JNKNF-kBOxidative StressRhoATGF-bWnt
Xenobiotic Stress
Activator/Pathway a
NFAT
CREBp53ATF6
HSF1MTF1Hif1αSTAT5:STAT5STAT3:STAT3STAT1:STAT2
Elk-1/SRFAP1NF-kBNrf2SRFSMAD3:SMAD4LEF-TCF
AhR
Transcription FactorE8481
E8471E3651E3661
E3751E4131E4001E4651E4041E4141
E1340E4111E8491E3641E1350E3671E4611
E4121
Catalog #pGL4.30[luc2P/NFAT-RE/Hygro]
pGL4.29[luc2P/CRE/Hygro]pGL4.38[luc2P/p53 RE/Hygro]pGL4.39[luc2P/ATF6 RE/Hygro]
pGL4.41[luc2P/HSE/Hygro]pGL4.40[luc2P/MRE/Hygro]pGL4.42[luc2P/HRE/Hygro]pGL4.52[luc2P/STAT5 RE/Hygro]pGL4.47[luc2P/SIE/Hygro]pGL4.45[luc2P/ISRE/Hygro]
pGL4.33[luc2P/SRE/Hygro]pGL4.44[luc2P/AP1 RE/Hygro]pGL4.32[luc2P/NF-kB-RE/Hygro]pGL4.37[luc2P/ARE/Hygro]pGL4.34[luc2P/SRF-RE/Hygro]pGL4.48[luc2P/SBE/Hygro]pGL4.49[luc2P/TCF-LEFRE/Hygro]pGL4.43[luc2P/XRE/Hygro]
pGL4 Signaling Vector NameNuclear Factor of Activated T-Cells(NFAT) Response ElementCyclic AMP Response Elementp53 Response Element (p53 RE)Activating Transcription Factor 6 Response Element (ATF6 ERSE)Heat Shock Element (HSE)Metal Regulatory Element (MRE)Hypoxia Response Element (HRE)STAT5 Response Elementsis-Inducible Element (SIE)Interferon Stimulated Response Element(ISRE)Serum Response ElementAP1 Response Element (AP1 RE)Nuclear Factor kB Response ElementAntioxidant Response Element (ARE)Serum Response Factor Response ElementSMAD Binding Element (SBE)TCF-LEF Response Element (TCF-LEF RE)
Xenobiotic Response Element (XRE)
Response Element
Firefly Luciferase Pathway Reporter
I . Firefly Luciferase Pathway Reporter Vectors and Stable Cell Lines
4
Promega offers its latest R&D reporter cell lines and vectors asCustom Assay Materials. These custom materials include differ-ent luciferases (firefly, Renilla or NanoLuc®) with varyingreporter stabilities (Luc2P, RlucP, and NlucP), and different selec-table markers (e.g., Hygromycin vs. Puromycin).
II. Custom Assay Materials—Reporter Vectors and Cell Lines
Custom Firefly Luciferase Pathway Reporter
Androgen Activation in Prostate CancerCytochrome P450/Drug MetabolismCytochrome P450/Drug MetabolismDNA Damage/p53ER Stress
HedgehogIL1JAK/STAT1 IFN-γJAK/STAT4 IL12MultipleMycNFATNGFNotchSTAT3T cell Activation, IL1T cell Activation, IL1TGF-b/BMPWnt
PathwaypGL4[luc2P/PSA-long/Hygro] Vector
pGL4[luc2P/3A4/Hygro] VectorpGL4[luc2P/2B6/Hygro] VectorpGL4[luc2P/p53-RE/Puro] VectorpGL4[luc2P/ATF4-RE/Hygro] Vector
pGL4[luc2P/Gli-RE/Hygro] VectorpGL4[luc2P/hIL8/Hygro] VectorpGL4[luc2P/GAS-RE/Hygro] VectorpGL4[luc2P/STAT4-RE/Hygro] VectorpGL4[luc2P/C/EBP-RE/Hygro] VectorpGL4[luc2P/Myc/Hygro] VectorpGL4[luc2P/NFAT-RE/Puro] VectorpGL4[luc2P/hEGR1/Hygro] VectorpGL4[luc2P/RBP-JK-RE/Hygro] VectorpGL4[luc2P/GCSF/Hygro] VectorpGL4[luc2P/IL-2/Hygro] VectorpGL4[luc2P/IL-2/Puro] VectorpGL4[luc2P/hID1/Hygro] VectorpGL4[luc2CP/TCF-LEF RE/Hygro]Vector
Vector NameHuman PSA Promoter
Human 3A4Human 2B6p53 Response Element (p53-RE)Activating Transcription Factor 4 Response Element (ATF4-RE)Gli Response Element (Gli)Human IL8 PromoterIFN-γ Activation Site (GAS) Response ElementIRF1 Response ElementC/EBP Response Element (C/EBP)Myc Response Element (Myc)NFAT Response ElementHuman Early Growth Response Factor-1(EGR 1) PromoterCBF-1/RBP-JK Response ElementHuman G-CSFHuman IL2 PromoterHuman IL2 PromoterHuman DNA-Binding Protein Inhibitor ID-1 PromoterTCF-LEF Response Element (TCF-LEF RE)
Response Element/Promoter
5
Calcium/CalcineurincAMP/PKADNA DamageER StressER StressHeavy Metal StressHypoxiaJAK/STAT1 IFN-γJAK/STAT1/2 IFN-αJAK/STAT3 IL-6JAK/STAT5 IL3MAPK/JNKMultipleNFkBOxidative StressPI3K/Akt/MAPKRas/MEK-1RhoA (Gα12/13)TGF-βWntXenobiotic Stress
PathwaypNL[NlucP/NFAT-RE/Hygro] VectorpNL[NlucP/CRE/Hygro] VectorpNL[NLucP/p53-RE/Hygro] VectorpNL[NlucP/ATF4-RE/Hygro] VectorpNL[NlucP/ATF6-RE/Hygro] VectorpNL[NlucP/MRE/Hygro] VectorpNL[NlucP/HRE/Hygro] VectorpNL[NlucP/GAS-RE/Hygro]d VectorpNL[NlucP/ISRE/Hygro]d VectorpNL[NlucP/SIE/Hygro] VectorpNL[NlucP/STAT5-RE/Hygro] VectorpNL[NlucP/AP1-RE/Hygro) VectorpNL[NlucP/CEBP-RE/Hygro] VectorpNL[NlucP/NFkB-RE/Hygro] VectorpNL[NlucP/ARE/Hygro] VectorpNL[NlucP/MycMax-RE/Hygro] VectorpNL[NlucP/SRE/Hygro] VectorpNL[NlucP/SRF/Hygro] VectorpNL[NlucP/SBE/Hygro] VectorpNL[NlucP/TCF/LEF-RE/Hygro]d VectorpNL[NlucP/XRE/Hygro] Vector
Vector NameNuclear Factor of Activated T-Cells (NFAT) Response ElementCyclic AMP Response Element (CRE)p53 Response Element (p53 RE)Activating Transcription Factor 4 Response Element (ATF4 ERSE)Activating Transcription Factor 6 Response Element (ATF6 ERSE)Metal Regulatory Element (MRE)Hypoxia Response Element (HRE)IFN-γ Activation Site (GAS) Response ElementInterferon Stimulated Response Element (ISRE)Sis-Inducible Element (SIE) Response ElementSTAT5 Response ElementAP1 Response Element (AP1 RE)CEBP Response ElementNuclear Factor kB (NFkB) Response ElementAntioxidant Response Element (ARE)Myc/Max Response ElementSerum Response Element (SRE)Serum Response Factor Response Element (SRF)SMAD3/SMAD4 Binding Element (SBE)TCF-LEF Response Element (TCF-LEF RE)Xenobiotic Response Element (XRE)
Response Element/Promoter
Custom NanoLuc Luciferase Pathway Reporter
II. Custom Assay Materials—Reporter Vectors and Cell Lines
Custom Renilla Luciferase Pathway Reporter
DNA damage/p53HypoxiaMAPK
PathwaypGL4[hRlucP/p53-RE/Hygro] VectorpGL4[hRlucP/HRE/Hygro] VectorpGL4[hRlucP/AP1 RE/Hygro] Vector
Vector Namep53 Response Element (p53-RE)Hypoxia Response Element (HRE)AP1 Response Element (AP1 RE)
Response Element/Promoter
6
Custom NanoLuc Luciferase Response Cell Lines
Custom Firefly Luciferase Response Cell Lines
AntioxidantCalcium/CalcineurinHypoxiaHypoxiaJAK/STAT1 IFN-γJAK/STAT1 IFN-γJAK/STAT3 IL6JAK/STAT3 IL6MAPKRas/MEK-1RhoA (Gα12/13)
PathwayHEK293JurkatHEK293HepG2HepG2HeLaHEK293HepG2HEK293HEK293HEK293
Cell Line pGL4.37[luc2P/ARE/Hygro] VectorpGL4[luc2P/NFAT-RE/Hygro] VectorpGL4.42[luc2P/HRE/Hygro] VectorpGL4.42[luc2P/HRE/Hygro] VectorpGL4[luc2P/GAS-RE/Hygro] VectorpGL4[luc2P/GAS-RE/Hygro] VectorpGL4.47[luc2P/SIE-RE/Hygro] VectorpGL4.47[luc2P/SIE-RE/Hygro] VectorpGL4.44[luc2P/AP1 RE/Hygro] VectorpGL4.33[luc2P/SRE/Hygro] VectorpGL4.34[luc2P/SRF-RE/Hygro] Vector
Reporter
II. Custom Assay Materials—Reporter Vectors and Cell Lines
NFkBPathway
HEK293Cell Line Background
pNL[NlucP/NFkB-RE/Hygro] VectorReporter
7
Let us help you choose!
We will gladly help you to find the right experimental and control vectors
for your experiment upon request. We will also provide you with the vector
maps and the GenBank/EMBL accession number of the appropriate vectors.
Request your customized vector or cell line!
Custom assay materials are not catalog products.
For ordering, please contact your Promega representative
www.promega.de/support/worldwide-contacts/
or visit www.promega.com/custom-assay
Stay A Step Ahead
Use the latest materials developed by Promega Custom Assay Services to
advance your discovery programs and keep ahead of the pack. Choose from
a range of vectors and cell lines based on proven, versatile luciferase
reporter systems. Visit our Custom Assay Materials web site for more infor-
mation about the Promega portfolio. www.promega.com/custom-assay
Can’t find what you are looking for? Contact our Technical Services Team to
find out all the options for a custom-made vector or cell line to advance
your research!
www.promega.de/support/customer-and-technical-support
Customer Support Telephone +49 621 8501-291Fax +49 621 [email protected]
Technical Support Telephone +49 621 [email protected]
Promega GmbH
High-Tech-ParkSchildkrötstraße 15 · 68199 MannheimTelephone +49 621 8501-0 · www.promega.com
