Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling

Protocol

Immunoblotting and sequential lysis protocols for the analysis of

tyrosine phosphorylation-dependent signaling

Caroline Gilberta,b, Emmanuelle Rollet-Labellea,b, Adriana C. Caonc,Paul H. Naccachea,b,*

aCentre de Recherche en Rhumatologie et Immunologie, CIHR group on the Molecular Mechanisms of Inflammation,

Centre de Recherche du CHUL, Laval University, Ste.-Foy, Quebec, CanadabDepartment of Medicine, Faculty of Medicine, Laval University, Ste.-Foy, Quebec, Canada

cDepartment of Molecular Biosciences, University of Adelaide, North Terrace, Adelaide, Australia

Received 30 August 2002; accepted 5 September 2002

Abstract

In stimulated neutrophils, the majority of tyrosine-phosphorylated proteins are concentrated in Triton X-100 or NP-40

insoluble fractions. Most immunobiochemical studies, whose objective is to study the functional relevance of tyrosine

phosphorylation are, however, performed using the supernatants of cells that are lysed in non-ionic detergent-containing buffers

(RIPA lysis buffers). This observation prompted us to develop an alternative lysis protocol. We established a procedure

involving the sequential lysis of neutrophils in buffers of increasing tonicities that not only preserve and solubilize tyrosine-

phosphorylated proteins but also retain their enzymatic activities. The sequential lysis of neutrophils in hypotonic, isotonic and

hypertonic buffers containing non-ionic detergents resulted in the solubilization of a significant fraction of tyrosine-

phosphorylated proteins. Furthermore, we observed in neutrophils in which CD32 was cross-linked that the tyrosine kinase

activity of Lyn was enhanced in the soluble fraction recovered from the hypertonic lysis but not in that derived from the first

hypotonic lysis. Furthermore, we detected tyrosine kinase activity and the presence of the tyrosine kinase Syk in association

with CD32 in the soluble hypertonic lysis fraction. This fraction also contained most of the tyrosine-phosphorylated proteins

including Cbl, Syk and CD32 itself. The results of this study provide a new experimental procedure for the investigation of

tyrosine phosphorylation pathways in activated human neutrophils which may also be applicable to other cell types.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Cbl; CD32; Detergent insolubility; Fcg receptors; Kinase activity; Lyn; Monosodium urate monohydrate crystals; Neutrophil;

Phagocytosis; Signal transduction; Syk; Tyrosine phosphorylation

1. Background

Neutrophils play a crucial role in host defense

against injury and infection as well as in the inflam-

matory response (Smith, 1994) by virtue of their

ability to mount a series of effector responses. Neu-

trophils respond to a wide variety of agonists and one

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00347 -2

Abbreviations: HLB, hypotonic lysis buffer; HyperLB, hyper-

tonic lysis buffer; ILB, isotonic lysis buffer; LB, lysis buffer; RIPA,

rapid immunoprecipitation assay; SB, sample buffer.

* Corresponding author. CHUL, Room T1-49, 2705 Boulevard

Laurier, Ste.-Foy, Quebec, Canada G1V 4G2. Tel.: +1-418-654-

2772; fax: +1-418-654-2765.

E-mail address: [email protected]

(P.H. Naccache).

www.elsevier.com/locate/jim

Journal of Immunological Methods 271 (2002) 185–201

of the earliest events observed upon stimulation of

neutrophils is an increase in the level of tyrosine

phosphorylation of a number of proteins (Rollet et

al., 1994).

The mechanisms linking neutrophil surface recep-

tors to tyrosine kinase signaling are, however, incom-

pletely understood. Immunoprecipitation coupled

with immunoblotting is one of the most commonly

used immunochemical techniques for studying tyro-

sine phosphorylation-dependent pathways. This tech-

nique can potentially determine several important

characteristics of molecules under examination such

as their presence and quantities, their relative molec-

ular weights, their rates of synthesis or degradation,

and the presence of certain post-translational mod-

ifications. It can also provide information about

interactions between proteins, nucleic acids and

other ligands. These protocols depend on lysing

the cells under mild conditions (usually RIPA buf-

fers) containing cocktails of protease and phospha-

tase inhibitors. These lysates are then centrifuged

and the supernatants are used for subsequent immu-

noprecipitations. It is commonly observed, however,

that the profile of tyrosine-phosphorylated proteins

of whole neutrophils is rapidly lost or artificially

increased upon lysing the cells in classical RIPA

buffers (Al-Shami et al., 1997b; Naccache et al.,

1997). An alternative assay involving lysis under

denaturing conditions was previously developed to

preserve the phosphorylation levels in neutrophil

lysates (Al-Shami et al., 1997b). Although this

protocol allows the identification of tyrosine-phos-

phorylated proteins (Al-Shami et al., 1997a; Barabe

et al., 1998; Khamzina and Borgeat, 1998; Rollet-

Labelle et al., 2000; Gilbert et al., 2001), it disrupts

protein–protein interactions and inactivates most

enzymatic activities.

Most studies analyze the soluble fractions fol-

lowing lysis in RIPA buffers while discarding the

insoluble fractions. However, the formation of

detergent-resistant membrane structures (DRM,

DIGs or lipids rafts) into which tyrosine-phosphory-

lated proteins and tyrosine kinases tend to concen-

trate after receptor stimulation has been well-

described in a range of cell types (Brown and

London, 1998; Simons, 2000) including neutrophils

(Zhou et al., 1995; Yan et al., 1996; Barabe et al.,

2002). The function of these detergent-insoluble

structures appears to be related to signal trans-

mission and/or membrane trafficking. Accordingly,

tyrosine kinase activities were found to be increased

and concentrated in these insoluble fractions upon

ligation of phagocytic receptors in adherent human

neutrophils (Zhou et al., 1995; Yan et al., 1996).

Accurate analysis of tyrosine phosphorylation

events in these structures depends on the prepara-

tion of stable cell lysates that can be used as

starting material for their analysis by immunopreci-

pitation.

In the present study, a method designed to reach

the above aims, based on sequential cell lysis under

conditions of increasing tonicity, is described. Firstly,

we show the distribution and preservation of tyrosine-

phosphorylated substrates in human neutrophil lysates

prepared under native conditions. The results obtained

illustrate the inadequacy of using standard cell lysates

prepared under native conditions as starting material

in subsequent immunoprecipitation or co-immunopre-

cipitation protocols. Secondly, we observe, using our

modified protocol, that the tyrosine kinase activity of

Lyn is specifically increased in the hypertonic lysis

fractions. This fraction is enriched in GM1 ganglio-

side and cytoskeletal components. Thirdly, we show

in the hypertonic lysis fractions an association

between the tyrosine kinase Syk and cross-linked

CD32. Finally, we also observe that Cbl, Syk and

CD32 were highly tyrosine-phosphorylated, specifi-

cally in the soluble fraction of the hypertonic lysis

step. The use of this alternative protocol emphasizes

the critical importance of the complete monitoring of

the tyrosine phosphorylation status as well as of the

solubility and, when applicable, the enzymatic activ-

ity of each target molecule, in response to each

agonist.

2. Type of research

(i) Signal transduction.

(ii) Analysis of tyrosine phosphorylation profiles and

identification of tyrosine-phosphorylated sub-

strates.

(iii) Distribution of tyrosine-phosphorylated proteins

between soluble and insoluble fractions.

(iv) Measurements of tyrosine kinase activities upon

stimulation in the soluble and insoluble fractions.

C. Gilbert et al. / Journal of Immunological Methods 271 (2002) 185–201186

3. Time required

(i) Cell isolation (2 h)

(ii) Cell stimulation and lysis (15–45 min)

(iii) Lysate immunoprecipitation (3 h)

(iv) Kinase activity (10–60 min)

(v) Electrophoresis (5 h or O/N)

(vi) Transfer to Immobilon PVDF membranes (3 h or

O/N)

(vii) Immunoblot (3–4 h).

4. Materials

4.1. Preparation of cells

HBSS 1� solution (Wisent Canadian Laborato-

ries, St.-Bruno, Quebec, Canada) containing 1.6

mM CaCl2 + 10 mM HEPES

2% of dextran solution (Pharmacia Biotech,

Dorval, Quebec, Canada in HBSS 1� )

Ficoll Paque (Wisent Canadian Laboratories)

10% trypan blue solution (Sigma-Aldrich Canada,

Oakville, ON, Canada).

4.2. Preparation of buffers

2� Laemmli’s sample buffer (2� SB)

125 mM Tris–HCl, pH 6.8

8% SDS

10% h-mercaptoethanol

17.5% glycerol

5 mM Na3VO4 (stock solution is prepared

as previously described (Papavassiliou,

1994))

20 mM p-nitrophenylphosphate

20 Ag/ml leupeptin

20 Ag/ml aprotinin

0.0025% bromophenol blue

2� lysis buffer (2�LB)

125 mM Tris–HCl, pH 6.8

4% SDS

3% h-mercaptoethanol

17.5% glycerol

5 mM Na3VO4

20 Ag/ml leupeptin

20 Ag/ml aprotinin

0.0025% bromophenol blue

Hypotonic lysis buffer (HLB)

0.1% NP-40 (10% NP-40, Calbiochem, La Jolla,

CA, USA)

20 mM Tris–HCl, pH 7.5 at 4 jC10 mM NaCl

1 mM EDTA

2 mM Na3VO4

10 Ag/ml aprotinin

10 Ag/ml leupeptin

2 mM PMSF (optional, to be added immedi-

ately prior to lysing the cells)

50 Ag/ml soybean trypsin inhibitor

3 mM DFP (to be added just prior to lysing the

cells. Take care to discard the tips in a tube

containing 1N NaOH in order to neutralize the

DFP)

Isotonic lysis buffer (ILB)

1% NP-40 (100% NP-40, Calbiochem)


1 mM EDTA

2 mM Na3VO4

10 Ag/ml aprotinin

10 Ag/ml leupeptin

2 mM PMSF


Hypertonic lysis buffer (HyperLB)

1% NP-40 (from 100% NP-40, Calbiochem)


1 mM EDTA

2 mM Na3VO4

10 Ag/ml aprotinin

10 Ag/ml leupeptin

2 mM PMSF


Immunoprecipitation wash buffer (IPPWashB)

1% NP-40 (from 100% NP-40, Calbiochem)


1 mM EDTA (omit in the last wash if measuring

kinase activity)

Kinase buffer (1�KB)

50 mM HEPES, pH 7.5

10 mM MgCl23 mM MnCl250 AM ATP (prepared from powder just prior to

starting the kinase assay)

C. Gilbert et al. / Journal of Immunological Methods 271 (2002) 185–201 187

0.5 Ag of SAM68–GST per immunoprecipita-

tion.

4.3. Preparation of polyacrylamide gel (7.5–20%

SDS-PAGE)

30% acrylamide solution

0.3% bisacrylamide solution

This provides optimal separation for a wide range

of molecular weight proteins (8–150 kDa).

4.4. Preparation of transfer and blotting solutions

Transfer buffer

25 mM of Tris base

192 mM glycine

20% (v/v) methanol

Ponceau 1%

1.0 g Ponceau S

1.0 ml glacial acetic acid

99.0 ml H2O

Tris buffer/Tween 20 (TBS)

25 mM Tris HCl, pH 8.0

190 mM NaCl

0.15% (v/v) Tween 20

Blocking solution

2% (v/v) gelatin

100 ml TBS

Microwave for 30 s to dissolve gelatin or warm

in a water bath or on a hot plate. Cool to 37 jCbefore use.

4.5. Antibodies

(1) The polyclonal antibodies, anti-Cbl (sc-170,

1:1000), anti-Lyn (sc-15, 1:2000) and anti-

SAM68 (sc-333, 1:1000) are purchased from

Santa Cruz Biotechnology (Santa Cruz, CA,

USA). The rabbit anti-mouse heavy/light chains

(315-005-048) is purchased from Jackson Im-

mune Research (Mississauga, ON, Canada).

(2) The monoclonal antibodies anti-Syk (MAB88906,

1:1000), anti-phosphotyrosine (UBI 05-321, clone

4G10, 1:4000) are purchased from Chemicon

International (Missisauga, ON, Canada) and

Upstate Biotechnology (Lake Placid, NY, USA),

respectively. The monoclonal antibodies anti-

Ezrin (E53020, 1:5000), anti-p62 nucleoporin

(N43620, 1:1000) and the organelle Sampler kit

antibodies (611436) are obtained from Trans-

duction Laboratories (Lexington, KY, USA).

(3) An anti-FcgRII polyclonal antiserum against the

cytoplasmic tail of CD32 (CT-10) is generated in

our laboratory as described previously (Ibarrola et

al., 1997).

(4) Monoclonal anti-FcgRII antibodies (IV.3) are

purified from the ascites fluid of mice inoculated

with hybridoma HB 217, which is obtained from

the American Type Culture Collection (Rock-

ville, MD, USA). F(abV)2 fragments of the IV.3

antibody are prepared essentially as described in

the Pierce catalog (Rockford, IL, USA). Briefly,

the antibodies are digested with pepsin and

intact antibodies are eliminated by adding

protein A and protein G beads. The integrity

and purity of the F(abV)2 fragments are verified

by their ability to label intact human neutrophils

as determined by flow cytometry as well as by

their neutrophil-activating properties (calcium

mobilization, superoxide generation, tyrosine

phosphorylation).

4.6. Reagents

Soybean trypsin inhibitor, cholera toxin B-HRP

and sodium orthovanadate (Na3VO4) are purchased

from Sigma-Aldrich Canada. Di-isopropylfluorophos-

phate (DFP) (NP-40 10% and 100% solution) are

obtained from Calbiochem. p-Nitrophenylphosphate,

aprotinin and leupeptin are purchased from ICN

Pharmaceuticals (Costa Mesa, CA, USA). Sephadex

G-10, protein A sepharose are purchased from Phar-

macia Biotech. SAM68–GST fusion protein is ob-

tained from Santa Cruz Biotechnology.

4.7. Special equipment

1. Centrifuge

2. Microcentrifuge

3. Thermomixer R at 37 and 30 jC (Eppendorf)

4. Dry bath at 100 jC5. Sonicator

6. Rotator platform at 4 jC7. Peristaltic pump

8. Electrophoresis slab gel apparatus

9. Electrophoretic Transfer Unit


10. Power supply

11. Rocking platform

12. Microcentrifuge tubes.

5. Detailed procedures

5.1. Cell preparation

Peripheral blood is obtained from healthy adult

volunteers and collected on isocitrate anticoagulant.

Neutrophil suspensions are prepared sterilely as pre-

viously described (Pouliot et al., 1998), except that

sedimentation of erythrocytes is performed using 2%

dextran solution diluted in HBSS. The cells are

resuspended at a concentration of 107 cells/ml in

HBSS, pH 7.4, containing 1.6 mM calcium but no

magnesium in order to minimize aggregation. Cell

preparations with non spherical morphologies (an

indication of pre-activation) are excluded from the

study.

5.2. Cell stimulation

Before all stimulations, neutrophil suspensions

(4� 107 cells/ml) are pre-incubated at room temper-

ature with 1 mM DFP for 5 min. Neutrophils suspen-

sions of the same concentration are then stimulated

with the desired agonists at 37 jC. To cross-link

CD32, the cell suspensions are incubated with 2.0

Ag/ml of anti-FcgRII (IV.3) antibodies for 1 min at 37

jC and then with 20 Ag/ml of F(abV)2 goat anti-mouse

Fc antibody (Jackson Immune Research) for 1 min at

37 jC or the indicated periods of time. For immuno-

precipitations, the F(abV)2 fragment of IV.3 is used

instead of the whole antibody.

5.3. Preparation of total cell lysates under denaturing

conditions

After stimulation, the reactions are stopped by

transferring 100 Al of the cell suspensions directly to

an equal volume of boiling 2� sample buffer. The

samples are boiled for 7 min and vortexed frequently

to ensure that the cells have dissolved properly. The

samples are loaded immediately on SDS polyacryla-

mide gels, or stored at � 80 jC (if frozen, it is best to

reheat samples before loading onto gels.). It is critical

to add the cells directly to boiling sample buffer as

this greatly reduces the background levels of tyrosine

phosphorylation due to nonspecific activation. The

addition of DFP (1–4 mM) to the resuspension buffer

helps minimize proteolysis and activation caused by

centrifugation (Gilbert et al., 2001). The conditions

outlined above should apply equally to other leuko-

cyte populations.

5.4. Hypotonic cell lysis

After stimulation, the reactions are rapidly stop-

ped by transferring the cell suspensions to precooled

(� 20 jC) 1.5 ml microcentrifuge tubes. The cells

are then centrifuged for 5–10 s at 6000� g in a

microcentrifuge and the pellets resuspended at a

final concentration of 4� 107 cells/ml in the hypo-

tonic lysis buffer (HLB). For better inhibition of

phosphatases, 10 mM p-nitrophenylphosphate may

be added to the lysis buffer. After a 5-min incuba-

tion at 4 jC, the lysates are centrifuged at 600� g

for 10 min at 4 jC. Supernatants generated can be

used either for immunoprecipitation under native or

denaturing conditions, or for the determination of

tyrosine kinase activities, or transferred to an equal

volume of boiling 2� SB. Similarly, the resulting

pellets are either resuspended in HLB and then

transferred to an equal volume of boiling 2� SB,

or ILB or HyperLB for immunoprecipitation or

determination of tyrosine kinase activity. Nuclear

integrity and total cell lysis are routinely verified

by light microscopy.

5.5. Isotonic cell lysis

After stimulation, the reactions are rapidly stopped

by transferring the cell suspensions into precooled

(� 20 jC) 1.5 ml microcentrifuge tubes. The cell

suspensions are then centrifuged for 5–10 s at

6000� g in a microcentrifuge. The supernatants are

removed and the cell pellets are resuspended and

incubated for 5 min at 4� 107 cells/ml at 4 jC in

cold isotonic lysis buffer (ILB). The lysates are

centrifuged at 600� g for 10 min at 4 jC. Aliquotsof the supernatants are added to an equal volume of

boiling 2� SB. The pellets are resuspended in ILB

and then transferred to an equal volume of boiling

2� SB.


5.6. Sequential solubilization

The soluble and insoluble fractions of the hypo-

tonic lysis are individually processed for subsequent

analysis. The soluble fraction is centrifuged at

180,000� g at 4 jC for 45 min. The supernatant is

analyzed by transfer of an aliquot into 2� SB. The

pellet is dissolved in 1 volume of HLB and transferred

to boiling 2� SB. The fraction insoluble in HLB is

resuspended in ILB and centrifuged for 10 min at

13,000� g. The soluble material of this second lysis

is transferred into 2� SB and the pellet is resuspended

in the hypertonic lysis buffer (HyperLB). Two 5-s

sonication steps may be necessary for maximal pellet

extraction depending on the individual sonicator used.

Some adjustment may be necessary to maximize

extraction and preservation of intact phosphorylated

products. The lysates are centrifuged at 13,000� g for

10 min at 4 jC. Aliquots of the supernatants of each

fraction (soluble HLB to soluble HyperLB) are trans-

ferred to an equal volume of boiling 2� SB. The

pellets are resuspended in HLB and then diluted in the

same volume of 2� SB. The samples are then electro-

phoresed as described above. The soluble fractions of

HyperLB are also used under native conditions for

immunoprecipitation and tyrosine kinase activity.

These fractions (soluble HyperLB) can also be

obtained by direct addition of HyperLB to the HLB

pellets. The experimental scheme illustrated in Fig. 1

shows the details of this procedure.

5.7. Immunoprecipitation under denaturing condi-

tions

Neutrophils are incubated and stimulated as

described above. Aliquots (500 Al) of the cells are

lysed by direct transfer to an equal volume of boiling

2� lysis buffer (2�LB) and boiled for 7 min.

Immunoprecipitations are performed as previously

described (Al-Shami et al., 1997b). Briefly, lysates

are filtered through sephadex G-10 columns to

remove the denaturing agents. The filtered lysates

are precleared with protein A-sepharose at 4 jC for

30 min in the presence of 1% NP-40, 2 mM orthova-

nadate, 10 Ag/ml leupeptin and 10 Ag/ml aprotinin.

The samples are then immunoprecipitated using 2 Agof anti-Cbl, 2 Ag of anti-Syk or 6 Ag of anti-CD32

previously bound to protein A-sepharose for 90 min at

4 jC on a rotator platform with constant end-over-end

mixing. The beads are collected and washed four

times with a lysis buffer containing 137 mM NaCl,

1% NP-40 but no SDS, h-mercaptoethanol or bromo-

phenol blue. Sample buffer (40 Al, 2� ) is added to

the beads which are then boiled for 7 min. The

proteins in the samples are then separated by electro-

phoresis as described above. The membranes are

blotted with anti-phosphotyrosine or with the immu-

noprecipitating antibodies (anti-Syk, anti-Cbl or anti-

CD32) for visualizing the amounts of precipitated

protein.

5.8. Native immunoprecipitation and tyrosine kinase

activity

The aliquots of HLB or HyperLB supernatant

lysates are mixed with HyperLB or HLB, respectively,

to bring back the NaCl and NP-40 concentrations to

137 mM and 1%, respectively. These isotonic lysates

are immunoprecipitated using 1.5 Ag of anti-Lyn or 3

Ag of rabbit anti-mouse heavy/light chain antibodies

(for CD32 immunoprecipitation) for 90 min at 4 jCon a rotator platform with constant end-over-end

mixing. The RAM antibodies recognize the anti-

CD32 fragment antibodies fixed on the cells during

stimulation and do not react with the goat anti-mouse

antibodies used for cross-linking CD32. Fifty micro-

liters (30% slurry) of protein A-sepharose is then

added and the samples are incubated for 1 h at 4

jC. The beads are collected and washed four times

with ILB buffer without EDTA. The beads are incu-

bated at 4 jC in kinase buffer and transferred to 37 jCfor the indicated times. The reactions are stopped by a

quick spin and the supernatants are discarded and then

2� SB is added to the beads. In the case of SAM68–

GST, when present in the KB mix, the supernatants

are precipitated with sepharose–GST beads and

washed twice with ILB buffer. Sample buffer (40 Al,2� ) is added to the beads which are then boiled for 7

min. The proteins in the samples are then separated by

electrophoresis as described above. The membranes

are blotted with the anti-phosphotyrosine or with the

specific immunoprecipitation antibodies for visualiz-

ing the amounts of precipitated protein. Immunopre-

cipitation with control rabbit IgG followed by kinase

activity is undertaken in parallel to verify the specif-

icity of the precipitations.


5.9. Detailed electrophoresis and immunoblotting

procedure

Prior to loading onto 7.5–20% SDS-PAGE acyla-

mide gels, samples in 2� SB are boiled for 7 min.

Proteins are then transferred to Immobilon PVDF

membranes (Millipore, Bedford, MA, USA). Gradient

gels (7.5–20%) and Immobilon PVDF membranes

have been found to be optimal for monitoring the

overall profile of tyrosine phosphorylation and for

effectively binding the majority of transferred pro-

teins. After transfer, the proteins are visualized with a

1% (w/v) Ponceau solution and the molecular weights

markers are identified. Nonspecific sites are blocked

with a 2% (w/v) gelatin solution for 30–60 min on a

rocking platform (not an orbital shaker). Dried milk

(i.e., Blotto) is not recommended as a blocking

solution for tyrosine phosphorylation immunoblotting

as it increases background. All subsequent incuba-

tions are performed on a rocking platform with con-

stant but not too vigorous rocking. We have found it

important to have only one membrane per container.

The blocking solution is discarded and replaced with a

2% gelatin solution containing a 1:4000 dilution of

anti-phosphotyrosine antibody (UBI 05 321) and

incubated for 1 h at 37 jC. The containers are rinsed

with water to remove any residual antibody solution

and the membranes are then washed with TBS 1�(three washes of 2 min each). The membranes are then

incubated with conjugated secondary antibody, either

goat anti-mouse-HRP antibodies ((#NXL-931)

1:20,000, Amersham/Pharmacia, Baie D’Urfe, QC,

Canada) or donkey anti-mouse-HRP antibodies

([#715-035-150] Jackson Immune Research) in 2%

gelatin solution or in TBS/Tween for 30–60 min at 37

jC or at room temperature equivalently. The secon-

dary antibody solution can only be used once and

should be made fresh for each assay. The containers

are rinsed with water and the membranes washed four

times for 5 min in a container with a large volume of

TBS/Tween (e.g., for a 12� 12 cm membrane, around

125 ml of TBS/Tween per wash). Care needs to be

taken when handling the membranes as any damage

can increase background staining. The detection sys-

tem solution is prepared as described in the data sheets

of the manufacturer (NEN Life Science, Boston, MA).

The membranes are placed protein facedown in a

clean container with the detection solution for 1 min

at room temperature without agitation. The solution is

dripped away for a few seconds and the membranes

are placed on a sheet of plastic food wrap large

enough to cover the membrane completely. The edges

are sealed and the membranes placed protein side up

in an autoradiography cassette. For clear and well-

focussed bands, the exposure of the film to the

membranes in the cassette should be conducted with-

out intensifying screens. For the other antibodies,

immunoblotting is performed using the appropriate

antibodies (anti-Cbl (1:1000), anti-CD32 (1:1000),

anti-SAM68 (1:1000) and anti-Syk (1:1000)) and

revealed using the Renaissance detection system

(NEN Life Science) using HRP-conjugated secondary

donkey anti-mouse [#715-035-150] or donkey anti-

rabbit [#711-035-152] antibodies (Jackson Immune

Research) at a dilution of 1:20,000 as described

above.

6. Results

6.1. Experimental schema of the sequential solubili-

zation protocol

Tyrosine phosphorylation in total neutrophil

lysates can be analyzed by immunoprecipitation

under denaturing conditions as illustrated in the left

side of Fig. 1. A major limitation of this technique

(Al-Shami et al., 1997b) is that it is impossible to

simultaneously monitor tyrosine kinase activity.

Aware of this limitation, we developed an alternate

protocol which preserved the tyrosine phosphoryla-

tion and the kinase activity and which is based on an

initial lysis in a hypotonic buffer (Gilbert et al.,

2002). The soluble and insoluble fractions of the

hypotonic lysis are therefore individually processed

for subsequent analysis as indicated in the right side

of Fig. 1.

We wanted to better characterize the soluble

fraction of the hypotonic lysis. After stimulation,

the soluble fraction of the hypotonic lysis is centri-

fuged for 45 min at 180,000� g. The resulting

soluble and insoluble fractions can be analyzed by

immunoblotting with anti-phosphotyrosine or other

antibodies.

Since most of the tyrosine-phosphorylated pro-

teins are recovered in the pellets following hypo-


tonic lysis, a further extraction of this pellet is

warranted in order to carry out immunoprecipita-

tions from this fraction. The HLB-insoluble fraction

is sequentially lysed in buffers of increasing tonic-

ity, the composition of which is described in

Section 4 (detailed procedures). Briefly, the HLB-

insoluble material is resuspended in ILB (dotted

line) or in HyperLB, and separated after centrifu-

gation into the soluble and insoluble fractions. The

ILB-insoluble material is then resuspended in

HyperLB, and soluble and insoluble fractions are

isolated after centrifugation. The relevant fractions

are analyzed by immunoblotting with anti-phospho-

tyrosine antibodies (Fig. 2, panel A), or immuno-

precipitated for the analysis of the translocation,

phosphorylation or kinase activity of various pro-

teins as illustrated in Fig. 3.

6.2. Effect of tonicity of the lysis buffer on the

preservation and distribution of tyrosine phosphory-

lation patterns and signaling-associated molecules

The difficulties associated with the preservation of

phosphotyrosine profiles and protein integrity in

human neutrophils have previously been described

(Al-Shami et al., 1997b; Naccache et al., 1997). The

extraction of tyrosine-phosphorylated proteins is

known to be affected by the composition of the lysis

buffer (Ignatoski and Verderame, 1996); the pH,

presence of phosphate, salt concentration, cell con-

centration, presence of cations, protease and phos-

phatase inhibitors can all modify the detergent

behavior and the solubilization of individual pro-

teins. Previous studies have reported the solubiliza-

tion of proteins without affecting the structure of the

Fig. 1. Experimental schema of sequential solubilization protocol. This schema represents several alternatives for studying the tyrosine kinase

pathway in the neutrophil. This procedure is also applicable for the other cells.


nuclei using a hypotonic lysis buffer coupled with

low concentrations of detergents (0.1% NP-40) (Pou-

liot et al., 1996; McDonald et al., 1998; Gilbert et

al., 2001, 2002). The data in Fig. 2 compare this

hypotonic buffer (HLB) with a more classical iso-

tonic RIPA buffer (ILB). The soluble and insoluble

fractions of the two lysis buffers (lanes 3–10) are

compared to the whole cell lysates (lanes 1–2). The

protein (Coomassie blue staining) and tyrosine phos-

phorylation profiles are illustrated in panels A and B

for the soluble (lanes 3–4, 7–8) and insoluble (lanes

5–6, 9–10) fractions derived from these cell lysis

protocols. To study the tyrosine phosphorylation

profile, neutrophils are stimulated by cross-linking

CD32 for 1 min at 37 jC as described in Section 4

(detailed procedure). The samples are analyzed by

Fig. 2. Distribution and preservation of tyrosine phosphorylation profiles in HLB and ILB. Neutrophils (4� 107 cells/ml) are stimulated by

cross-linking CD32 as indicated in the detailed procedure. The reactions are stopped by a transfer of cell aliquots into a precooled Eppendorf

tube followed by rapid centrifugation. The appropriate lysis buffer is then added to the cell pellet, HLB for lanes 3–6 and ILB for lanes 7–10 or

in the boiling 2� SB for lanes 1 and 2. Aliquots of soluble (lanes 3,4 and 7,8) and insoluble (lanes 5,6 and 9,10) fractions are diluted in an equal

volume of 2� SB. The samples are analysed by Coomassie blue protein staining (panel A) or by Western blot with anti-phosphotyrosine

(panel B).


Coomassie blue protein staining (panel A) or by

immunoblotting with anti-phosphotyrosine antibodies

(panel B), respectively.

The percentage of lysed cells and the integrity of

the nuclei are routinely verified by light microscopy

(data not shown) or by immunoblot analysis with

Fig. 3. Tyrosine kinase activity in the hypotonic and hypertonic soluble fractions. Neutrophils (4� 107 cells/ml) are stimulated by cross-linking

CD32 for 1 min at 37 jC as described in detailed procedure. The reactions are stopped by a transfer in a precooled Eppendorf tube followed by

rapid centrifugation. The HLB+ p-nitrophenylphosphate is added to the cell pellet. The aliquots (200 Al or 8� 106 equivalent cells) of the HLB

lysate supernatants are diluted with 200 Al of HyperLB and 200 Al of HLB. The pellet of the hypotonic lysis is directly incubated in a second

lysis buffer (HyperLB) and sonicated 2� 5 s. The soluble material (200 Al or 8� 106 equivalent cells) of this lysate are diluted in 2 volumes of

HLB and incubated with the anti-Lyn antibodies, and the kinase activity is performed as described in the detailed procedure. Aliquots of each

fraction are analysed by immunoblotting with an anti-phosphotyrosine antibodies in a panel A. Autophosphorylation is visualised by Western

blot with anti-phosphotyrosine antibody in a panel B.


nuclear markers such as p62 nucleoporin (a constitu-

ent of nuclear membranes) (Fig. 4). This confirmed

that the cells are completely lysed in the two buffers

and that nuclear integrity is better preserved in HLB

than in ILB (data not shown). Coomassie blue staining

(panel A) of these samples indicated that the amount

of protein extracted with the two lysis buffers is

roughly similar.

The tyrosine phosphorylation profile induced by

CD32 cross-linking, as monitored after the direct

transfer of the cells into sample buffer is illustrated

in the first two lanes of panel B and served as a

reference point for the evaluation of the adequacy of

the native cell lysates. Two major conclusions can be

drawn from monitoring the tyrosine phosphorylation

profiles in the various fractions (panel B). Firstly,

tyrosine-phosphorylated proteins are concentrated in

the insoluble fractions in both lysis protocols (lanes

5–6 and 9–10). Secondly, the tyrosine phosphoryla-

tion profiles are significantly better preserved in HLB

than in ILB (lanes 4–6 versus lanes 8–10).

Based on the three criteria, (i) prevention of pro-

teolysis (Gilbert et al., 2002), (ii) preservation of

tyrosine phosphorylation and (iii) day-to-day reprodu-

cibility of tyrosine phosphorylation levels (data not

shown), the data in Fig. 2 indicate that HLB is a

superior buffer for the preparation of starting material

from which to study tyrosine phosphorylation-

dependent signaling events in human neutrophils than

is ILB.

6.3. Sequential solubilization of the tyrosine-phos-

phorylated proteins

Since most of the tyrosine-phosphorylated pro-

teins are recovered in the pellets of the hypotonic

lysis, a further extraction of this pellet is warranted

in order to be able to carry out immunoprecipitation

from this fraction. On the other hand, it is important

to better characterize the soluble fraction of the

hypotonic lysis. The soluble and insoluble fractions

of the hypotonic lysis buffer are therefore individu-

ally processed for subsequent analysis. Fig. 3 shows

the anti-phosphotyrosine analysis of the soluble and

the insoluble fractions of the HLB (panel A) coupled

with the evaluation of the kinase activity of Lyn

(panel B).

After stimulation with the IV.3 F(abV)2, the solublefractions (lanes 3–4) of the hypotonic lysis are

conserved for immunoprecipitation with anti-Lyn

antibodies. The HLB-insoluble fraction is directly

lysed in HyperLB buffers and the soluble fractions

of this second lysis are conserved. The relevant

fractions are analyzed by immunoblotting with anti-

phosphotyrosine antibodies (panel A). The whole cell

lysates (lanes 1–2) served as the reference point for

the extent of stimulation as far as the global tyrosine

phosphorylation profile is concerned. As shown in

Fig. 3, the majority of the tyrosine-phosphorylated

proteins present in the pellet of the HLB are solubi-

lized and preserved in HyperLB (lanes 7–8) although

some tyrosine-phosphorylated proteins (lanes 9–10)

remained insoluble.

6.4. Lyn activity following its extraction from

HyperLB

In order to assess the state of the proteins

extracted in HyperLB, the immunoreactivity and

enzymatic activity of the Src kinase Lyn are tested

next in the soluble fractions of the HLB and

HyperLB lysates. It has previously been shown that

Lyn is activated in response to MSU crystals (Gau-

Fig. 4. Fraction characterization. Neutrophils (4� 107 cells/ml) are

lysed in 2�SB or in HLB buffer. The insoluble materials of the

HLB lysis are further processed in HyperLB buffer as described in

the detailed procedure. Aliquots of each fraction are diluted in

2� SB and analysed by Western blot with specific antibodies or

Cholera toxin HRP-coupled.


dry et al., 1995; Gilbert et al., 2002) and the

involvement of Lyn in activation via FcgRII has

also been described (Marcilla et al., 1995; Ibarrola et

al., 1997). To confirm this result following the

sequential lysis described here, neutrophils are stimu-

lated by pre-incubation with IV.3 F(abV)2 fragments

followed by addition of GAM for 1 min before being

lysed in HLB. HLB-soluble and -insoluble fractions

are prepared as described above. As the HLB-insolu-

ble material is also insoluble in ILB, the HLB-

insoluble fractions are directly resuspended in

HyperLB. Soluble and insoluble fractions are pre-

pared and Lyn is immunoprecipitated from the HLB-

and HyperLB-soluble fractions. The results shown in

Fig. 3B demonstrate that Lyn immunoprecipitated

from the HyperLB-soluble fractions retains in vitro

kinase activity. Furthermore, a stimulatory effect of

CD32 ligation on the auto-kinase activity of Lyn is

only detectable in the HyperLB lysates (Fig. 3B,

right side). Reblots indicate that equal amounts of

Lyn are immunoprecipitated and loaded in the con-

trol and CD32-stimulated cells (data not shown).

6.5. Characterization of the fractions

A partial characterization of the fractions obtained

during the sequential lysis protocol followed herein is

then carried out. Cytosolic markers (myeloid-related

protein-14 (MRP-14) and lactate dehydrogenase

(LDH)) are found predominantly in the soluble frac-

tion of the first lysis with HLB and in the soluble

fractions of the ultracentrifugation steps. Of relevance,

the receptor for cholera toxin (GM1) (Hart, 1975), a

major constituent of lipid rafts, is totally insoluble in

HLB buffer but is solubilized in HyperLB (Fig. 4).

Early endosome-associated protein-1 (EEA-1), an

endosomal marker, is equally distributed between

the soluble and insoluble fractions of HLB. The

HyperLB step completely solubilized EEA-1. Lyso-

some-associated membrane protein-1 (LAMP-1) is

entirely recovered in the HLB soluble fraction. Cytos-

keletal markers are also analyzed. A significant trans-

location of actin to the insoluble fraction is evident

after CD32 cross-linking as monitored by Coomassie

blue staining (Fig. 2). Paxillin and ezrin are equally

distributed between the soluble and the insoluble

fractions of the hypotonic lysis (Fig. 4). Extraction

with the hypertonic lysis buffer solubilized these

proteins. Finally, nuclear markers (p62 nucleoporin)

remain insoluble and are found in the pellets of the

HyperLB step (Fig. 4).

6.6. Association of kinase activity and Syk with CD32

precipitated in the HyperLB fractions

Stimulation of CD32 by cross-linking led to its

partitioning in the insoluble fraction in non-ionic

detergents, and, subsequently, its degradation (the

latter being reduced in the presence of tyrosine kinase

inhibitors (Barabe et al., 2002)). To confirm the

association of tyrosine kinase with CD32, CD32 is

cross-linked for increasing periods of time, the cell

pellets are lysed in HLB buffer and the insoluble

fractions are submitted to a second lysis in the

HyperLB as described above. The soluble material

of HyperLB fraction is incubated with 3 Ag of rabbit

anti mouse heavy/light chain antibodies and processed

for immunoprecipitation of CD32. Control experi-

ments established that the anti-CD32 antibodies fixed

on the cell by stimulation before lysis are preserved in

the presence of high salt concentrations and following

the sonication step. The immunoprecipitates are incu-

bated in a kinase buffer containing ATP and GST–

SAM68 fusion protein (as an exogenous substrate) for

10 min at 37 jC. The GST-fusion protein are recov-

ered with sepharose–GST beads and the CD32 immu-

noprecipitates are denaturated in 2� SB before

analysis by immunoblots (details in Section 4).

Immunoblots with an anti-phosphotyrosine anti-

body (Fig. 5, panel A) show a time-dependent asso-

ciation of tyrosine-phosphorylated proteins with the

CD32 immunoprecipitates. Two prominent bands

with migration characteristics that correspond to those

of CD32 and of the tyrosine Syk are consistently

detected. It should be pointed out that Src kinases are

masked by interference by the heavy chains of the

immunoprecipitating RAM antibodies. Association of

tyrosine kinase Syk with ITAM receptor has been

described upon CD32, CD64 and TCR activation

(Marcilla et al., 1995; Daeron, 1997; Bonnerot et

al., 1998). Immunomunoblot with anti-Syk antibodies

(panel B) reveal that the tyrosine kinase Syk is

present in the major tyrosine-phosphorylated band at

66 kDa observed in a panel A, and that this associ-

ation increased after CD32 ligation in a time-depend-

ent manner. Immunoblot with the anti-CD32 in panel


C shows that the amount of immunoprecipitated

CD32 is approximately equal. The results in panels

D and E confirm the presence of active tyrosine

kinase(s)-associated with CD32. Immunoblot of

GST–SAM68 precipitated with the anti-phosphotyr-

osine antibodies revealed that the CD32-associated

kinases are able to phosphorylate SAM68–GST

within 1 min of ligation, and that this phosphorylation

Fig. 6. Immunoprecipitation of tyrosine-phosphorylated proteins

from the HLB soluble and insoluble fractions. Neutrophils (4� 107

cells/ml) are stimulated by cross-linking CD32 as indicated in

Section 4. The reactions are stopped by a transfer of cell aliquots in

a precooled Eppendorf tube followed by rapid centrifugation. The

cell pellets are resuspended in HLB and the soluble and insoluble

fractions are diluted by 1/2 in boiling 2� LB. Immunoprecipita-

tions under denaturing conditions are performed as described in the

detailed procedure with the anti-Cbl, anti-Syk or anti-C32 anti-

bodies. Phosphorylated proteins are revealed with an anti-

phosphotyrosine antibody (upper panels), and the amount of

immunoprecipitated protein is visualized by immunoblot with the

respective antibodies (lower panels).

Fig. 5. Association of kinase Activity and Syk with the CD32

precipitated in the HyperLB fractions. Neutrophils (4� 107 cells/

ml) are stimulated by cross-linking CD32 for the indicated time at

37 jC as described in the detailed procedure. The reactions are

stopped by a transfer in a precooled Eppendorf tube followed by

rapid centrifugation. The cells are lysed with the HLB. The pellet of

the hypotonic lysis is directly incubated in a second lysis buffer

(HyperLB) and sonicated 2� 5 s. The soluble material (400 Al or16� 106 equivalent cells) of this lysis are diluted in 2 volumes of

HLB and incubated with 3 Ag of rabbit anti-mouse heavy/light chain

antibodies, and the immunoprecipitation and kinase activity assays

are performed as described in the detailed procedure. Kinase activity

associated with the CD32 is visualized by immunoblot with an anti-

phosphotyrosine antibodies (panel A). Protein associated with the

CD32 is identified in panel B by immunoblotting with an anti-Syk

antibodies. Amount of CD32 immunoprecipitated is evaluated in a

panel C with an anti-CD32 antibodies. Activity of associated

tyrosine kinases is verified in SAM68–GST precipitates as shown

in panels D and E by immunoblot with an anti-phosphotyrosine

antibodies (panel D) and anti-SAM68 (panel E).


is maintained for least 10 min. Immunoblot with the

SAM68 antibodies revealed that equal amounts of

GST–SAM68 are precipitated and loaded onto each

lane.

6.7. Tyrosine-phosphorylated proteins are concen-

trated in the insoluble fractions

The results described above revealed a heteroge-

nous distribution of phosphorylated proteins among

the soluble and insoluble fractions derived from the

lysis protocols after CD32 engagement. We therefore

monitored more closely three major tyrosine-phos-

phorylated substrates previously identified following

CD32 cross-linking, namely, Cbl, Syk and CD32 itself

(Rollet-Labelle et al., 2000) (Fig. 6). Briefly, aliquots

of the soluble fractions derived from the lysis of

control and CD32-stimulated cells in HLB are resus-

pended in an equal volume of boiling 2�LB as

described in above. The insoluble material is resus-

pended in the same volume of HLB as the soluble

fractions and subsequently in an equal volume of

boiling 2�LB. Cbl, Syk and CD32 are immunopre-

cipitated from both fractions. The precipitates are

probed with antiphosphotyrosine antibodies as well

as with anti-Cbl, anti-Syk or anti-CD32 antibodies

(panels A, B and C, respectively). The results obtained

show that phosphorylated Cbl, Syk and CD32 are

highly concentrated in the insoluble fractions in agree-

ment with the observations of Figs. 3 and 5.

Reblots with anti-Cbl, anti-Syk or anti-CD32 anti-

bodies (lower panels) indicate that cross-linking of

CD32 did not grossly modulate the amounts of Cbl

and Syk present in each fraction. On the other hand, a

significant decrease in the level of CD32 is observed

in the soluble fractions, and this is compensated for, in

part at least, by an increase in the level of CD32 in the

insoluble fractions.

7. Discussion

The protocols described above provide a frame-

work for the immunobiochemical investigation of

tyrosine phosphorylation-dependent signaling path-

ways in human neutrophils. The basis of this method

is an initial lysis in a hypotonic buffer. Under these

conditions, the overall tyrosine phosphorylation pro-

file is preserved to a major extent. The two fractions

thus obtained (soluble and insoluble) can then be

further analyzed, either directly (in the case of the

soluble fractions) or following solubilization in buf-

fers of increasing tonicities (in the case of the original

insoluble fractions). Each fraction can be analyzed by

immunoblotting or by monitoring enzymatic activ-

ities.

A major characteristic of these protocols is that

they allow retention of the tyrosine phosphorylation

profile, maintain enzymatic activities and preserve

associations with surface receptors. This procedure

also established that the detergent solubility of indi-

vidual tyrosine-phosphorylated substrates differed,

not only according to the protein under investigation,

but also in response to the specific agonist used

(Gilbert et al., 2002).

The severe experimental problems associated with

the preparation of neutrophil lysates (proteolysis, de-

phosphorylation or hyperphosphorylation) are widely

acknowledged but poorly documented. A previous

attempt to preserve the original patterns of tyrosine

phosphorylation relied on a denaturing lysis protocol

which, while effective, eliminated the possibility of

studying protein–protein interactions and enzymatic

activities (i.e., kinases and phosphatases) in the lysates

(Al-Shami et al., 1997b). The sequential lysis protocol

described in the present manuscript overcomes these

limitations in that the lysates prepared under native

conditions maintained to a significant extent their

profile of tyrosine phosphorylation (Fig. 2) as well

as their enzymatic activities (Figs. 3–5) and protein–

protein associations with surface receptor (Fig. 5).

Furthermore, whereas the stimulation of tyrosine

phosphorylation is transient in intact neutrophils, we

observed that the tyrosine phosphorylation in the

insoluble fractions was maintained for up to 60 min

following cell lysis. This can be explained by the

technique used to stop the stimulation, i.e., transfer of

the cells in precooled tubes followed by rapid cen-

trifugation. This approach immediately eliminates the

incubation medium which may contain degradative

enzymes (proteases) and phosphatases released by the

cells during stimulation.

One of the main findings of the present investiga-

tion was that the detergent extractability of the tyro-

sine-phosphorylated substrates was both substrate- and

agonist-dependent (Gilbert et al., 2002). Nevertheless,


a large percentage of the tyrosine-phosphorylated

proteins was insoluble in regular RIPA buffers of

hypo- or iso-tonicity (Fig. 2). This point raises ques-

tions about the interpretation of immunoprecipitation

studies in which the fraction that is soluble in these

lysis buffers is used as starting material in the absence

of a detailed picture of the distribution of the protein

being examined. Furthermore, the level of phosphor-

ylation of various proteins can be artifactually

increased by the presence of Mg2 + in the lysis buffers

(Gilbert et al., 2002), a procedure sometimes adopted

to help preserve membrane and cytoskeletal integrity.

Once again, this may impact on the interpretation of

the functional significance of apparent stimulation, or

lack of stimulation, of the tyrosine phosphorylation of

specific substrates in those cases where the profiles of

tyrosine phosphorylation of the lysates used as starting

material for the immunoprecipitation differs signifi-

cantly from that of whole cells.

The importance of knowing the distribution of

proteins in detergent extracts of the cells is strikingly

illustrated by the behavior of CD32, Cbl and Syk in

response to stimulation by CD32 ligation. All three

proteins were rapidly tyrosine-phosphorylated follow-

ing CD32 cross-linking (Marcilla et al., 1995; Nacc-

ache et al., 1997; Rollet-Labelle et al., 2000) and, as

shown in Fig. 6, they were recovered in both the

soluble and the insoluble fractions. However, in all

three cases, the tyrosine-phosphorylated fractions of

these proteins were highly concentrated in the deter-

gent-insoluble fractions. The physiological relevance

of the soluble form of these proteins is therefore likely

to differ from that present in the insoluble fraction.

Cross-linking of CD32 induces its insolubility in non-

ionic detergents, thereby implicating a potential role

for detergent-resistant membranes (Barabe et al.,

2002; Gilbert et al., 2002). Insolubility of membrane

receptors coupled to kinase activities were also

observed in T cell lines (Solomon et al., 1998) and

in various others cells (Zhou et al., 1995). In the case

of the stimulation of CD32, we were able to detect not

only its insolubility (Barabe et al., 2002; Gilbert et al.,

2002) (Figs. 5 and 6) but also the appearance of

tyrosine kinase activity that rapidly (after 1 min of

ligation) associated with CD32 in the insoluble frac-

tion of the HLB. Association of Syk with ITAM

motifs after tyrosine phosphorylation by Src family

kinase is a central feature of the classical models for

ITAM-motif activation. The results obtained in Fig. 5

show that our procedures allowed a time-dependent

association of Syk with CD32 to be detected (panel

B). We also observed the tyrosine phosphorylation of

a prominent band with electrophoretic migration char-

acteristics of Syk when we incubated the precipitated

cross-linked CD32 in a kinase buffer (panel A).

Pretreatment of the cells with the Src inhibitor PP-2

abolished this association (data not shown) in accord-

ance with the classical model of ITAM activation

described by others (Marcilla et al., 1995; Daeron,

1997; Bonnerot et al., 1998).

The functional relevance of the above considera-

tions is also highlighted by the results of the experi-

ments in which the kinase activity of Lyn was

monitored in the soluble fractions of both the hypo-

tonic and hypertonic lysis buffers (Fig. 3). The

enzyme remained active in both fractions, an indica-

tion of preservation of structure and function in the

lysates. Of more functional relevance, however, was

the observation that the stimulatory effect of CD32

cross-linking on the activity of Lyn was only readily

detectable in the hypertonic buffer lysate. A study of

the soluble fraction of the hypotonic lysate would

have missed this effect, while a lysis in an isotonic

buffer would be likely to result in a significant level

of signal distortion (proteolysis, dephosphorylation).

The sequential lysis protocol characterized in the

present study overcomes several of these problems

by preserving the original phosphorylation status and

by sequentially giving access to the various fractions.

This observation indicates that both fractions must

always be examined in these kinds of studies. These

data are consistent with the results of Zhou et al.

(1995), who previously reported a partitioning of Src

kinase activities between in detergent insoluble frac-

tions derived from adherent neutrophils. These en-

zymes are involved in the early steps of several

neutrophil responses and are differentially regulated

depending of their intracellular distribution (Welch

and Maridonneau-Parini, 1997). Our results also indi-

cate that, depending on the agonist used, the distri-

bution of the tyrosine-phosphorylated substrates

varies and must be individually characterized. Fur-

thermore, the addition of cofactors in the lysis buffer

must be carefully controlled.

In conclusion, we have demonstrated that the

detergent-insoluble fractions cannot be excluded in


biochemical studies of stimulated human neutrophils.

The concentration of signaling molecules in these

fractions may explain some of the controversial results

concerning tyrosine-phosphorylated proteins, tyrosine

kinase activities or protein associations that have been

reported in stimulated neutrophils. The present ap-

proach, which is based on the examination of all cell

fractions, may help to understand the biochemical

mechanisms regulating the functional responsiveness

of neutrophils. This method may also be extended to

the monitoring of phosphatase activities if Na3VO4 is

omitted from the lysis buffers allowing the evaluation

of the balance of kinase and phosphatase activities in

stimulated cells, which is most likely to be a key to the

regulation of several functional responses in many

cells.

8. Essential literature references

Al-Shami, A., Gilbert, C., Barabe, F., Gaudry, M.,

Naccache, P.H., 1997b. Preservation of the pattern of

tyrosine phosphorylation in human neutrophil lysates.

J. Immunol. Methods 202, 183–191.

Gilbert, C., Rollet-Labelle, E., Naccache, P.H.,

2002. Preservation of the pattern of tyrosine phos-

phorylation in human neutrophil lysates: II. A sequen-

tial lysis protocol for the analysis of tyrosine

phosphorylation-dependent signalling. J. Immunol.

Methods 261, 85–101.

Ignatoski, K.M., Verderame, M.F., 1996. Lysis

buffer composition dramatically affects extraction of

phosphotyrosine-containing proteins. BioTechniques

20, 794–796.

Welch, H., Maridonneau-Parini, I., 1997. Lyn and

Fgr are activated in distinct membrane fractions of

human granulocytic cells. Oncogene 15, 2021–2029.

Yan, S.R., Fumagalli, L., Berton, G., 1996. Acti-

vation of SRC family kinases in human neutrophils.

Evidence that p58C-FGR and p53/56LYN redistrib-

uted to a Triton X-100-insoluble cytoskeletal frac-

tion, also enriched in the caveolar protein caveolin,

display an enhanced kinase activity. FEBS Lett. 380,

198–203.

Zhou, M.J., Lublin, D.M., Link, D.C., Brown,

E.J., 1995. Distinct tyrosine kinase activation and

Triton X-100 insolubility upon Fc gamma RII or Fc

gamma RIIIB ligation in human polymorphonuclear

leukocytes. Implications for immune complex acti-

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Acknowledgements

Supported in part by grants from the Canadian

Institutes of Health Research. C. Gilbert is supported

by fellowships from the K.M Hunter Charitable

Foundation, the Canadian Institutes of Health Re-

search and the Fonds pour la Formation de Cher-

cheurs et l’Aide a la Recherche and the Fonds de la

Recherche en Sante du Quebec.

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Immunoblotting and sequential lysis protocols for the analysis of tyrosine phosphorylation-dependent signaling