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Analyzing FAK and Pyk2 in Early Integrin Signaling Events

Joie A. Bernard‐Trifilo1,  Ssang‐Taek Lim1,  Shihe Hou1,  David D. Schlaepfer1,  Dusko Ilic2

1The Scripps Research Institute, La Jolla, California
2Stem Life Line, Inc., San Carlos, California


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 14.7
DOI: 
10.1002/0471143030.cb1407s30
Online Posting Date: 
April, 2006
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Abstract

Integrins are a family of heterodimeric / transmembrane cell adhesion receptors that play important roles in the regulation of cell migration, proliferation, and survival. Integrins do not possess intrinsic catalytic activity, and signaling events are mediated by their lateral association with other cell surface receptors or clustering of their cytoplasmic domains with signaling proteins. Rapid activation of protein-tyrosine kinases is one of the first signaling events associated with integrin binding to the extracellular matrix protein fibronectin. The intracellular focal adhesion kinase (FAK) is recruited to sites of integrin clustering, and this unit describes the methods with which to analyze FAK phosphorylation, activity, and localization within fibroblasts. Additional methods on how to grow primary FAK+/+ and FAK–/– fibroblasts and measure integrin-stimulated cell motility are described as well as methods for evaluating the activity of the FAK-related kinase, Pyk2, which is expressed in FAK–/– cells.

Keywords: FAK; Pyk2; Src; tyrosine phosphorylation; focal adhesions; cell motility

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Replating Assays for Signaling Studies
  • Support Protocol 1: Growth of FAK+/+ and FAK–/– Fibroblasts
  • Support Protocol 2: Serum Starvation of Cells
  • Support Protocol 3: Preparation of Cell Lysates
  • Support Protocol 4: Immunoprecipitation of FAK, Pyk2, and c-Src
  • Basic Protocol 2: In Vitro Kinase Assay
  • Alternate Protocol 1: FAK-PYK2 Poly Glu:Tyr Phosphorylation
  • Alternate Protocol 2: Measurements of Src-Associated Kinase Activity
  • Basic Protocol 3: Immunoblotting with FAK/PYK2 Phospho-Specific Antibodies
  • Basic Protocol 4: Haptotaxis Motility Assay: Matrix-Stimulated Migration
  • Alternate Protocol 3: Analyzing Cell Motility Using Plasmid-Transfected Cells
  • Basic Protocol 5: Scratch-Wound Healing Assay With Time-Lapse Imaging
  • Basic Protocol 6: Immunolocalization of Focal Adhesion Proteins
  • Alternate Protocol 4: Immunolocalization of Actin Stress Fibers
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Replating Assays for Signaling Studies

 Materials
  • Fibronectin (from bovine plasma; Sigma-Aldrich)
  • Poly-l-lysine (mol. wt. 70,000 to 150,000 or 150,000 to 300,000; Sigma-Aldrich)
  • Replating and migration medium (see recipe)
  • FAK+/+ and FAK–/– MEFs, serum-starved (Support Protocol 2)
  • Phosphate buffered saline (PBS; appendix 2A)
  • Trypsin/EDTA: 0.25% (w/v) trypsin/1 mM EDTA (Invitrogen)
  • Trypsin inhibitor solution (see recipe)
  • 10-cm plastic tissue culture plates (Falcon)
  • 4°C incubator
  • 15-ml centrifuge tubes (Corning)
  • Tabletop centrifuge
  • 10-ml transfer pipets, sterile
  • 50-ml conical centrifuge tubes (Corning)
  • Light microscope

Support Protocol 1: Growth of FAK+/+ and FAK–/– Fibroblasts

 Materials
  • Gelatin
  • Phosphate buffered saline (PBS; appendix 2A)
  • FAK+/+ mouse embryo fibroblasts (ATCC CRL-2645) FAK–/– mouse embryo fibroblasts (ATCC CRL-2644): grown on plates as per ATCC product sheet
  • Trypsin/EDTA: 0.25% (w/v) trypsin/1 mM EDTA (Invitrogen), 37°C
  • Cell growth medium (see recipe), warmed to 37°C
  • 42°C water bath
  • 0.22-µm GP Express Plus filter membrane (Millipore)
  • 10-cm plastic tissue culture plates (Falcon)
  • 10-ml transfer pipets, sterile
  • Light microscope
  • 15-ml conical centrifuge tubes (Corning)

Support Protocol 2: Serum Starvation of Cells

 Additional Materials (also see Support Protocol 1)
  • Starvation medium: prepared by making cell growth medium (see recipe) with FBS reduced to 0.5%.

Support Protocol 3: Preparation of Cell Lysates

 Materials
  • Experimentally treated, control adherent, and control suspended cells (Basic Protocol 1)
  • Phosphate-buffered saline (PBS; appendix 2A), 4°C
  • RIPA cell lysis buffer (see recipe), 4°C
  • 50% (w/v) Sephadex G-100 (Sigma G100-120) slurry in PBS (appendix 2A)
  • Cell scraper (Corning)
  • Refrigerated centrifuge
  • 1.5-ml microcentrifuge tubes
  • 1-ml disposable syringes
  • 21-G Luer-Lok needles
  • Tube rotation device (e.g., Labquake, Barnstadt-Thermolyne)
  • Microcentrifuge
  • –80°C freezer (optional)

Support Protocol 4: Immunoprecipitation of FAK, Pyk2, and c-Src

 Materials
  • Primary antibody (see Table 14.7.1 and Table 14.7.2)
  • Lysate of treated or control cells (Support Protocol 3)
  • 50% (w/v) protein A-agarose beads (fast flow immobilized protein A; Repligen, http://www.repligen.com) in PBS (appendix 2A)
  • 50% (w/v) protein G-Plus-agarose beads (Calbiochem) in PBS (appendix 2A)
  • Triton lysis buffer (see recipe), 4°C
  • HNTG buffer (see recipe), 4°C
  • 1.5-ml microcentrifuge tubes
  • Tube rotation device (e.g., Labquake, Barnstadt-Thermolyne)
  • Centrifuge
  • Transfer pipets
     
    Table 14.7.1 Commercially Available Antibodies to FAK, Pyk2, and c-Srca
    AntibodyCompanySourceApplicationCatalog number
    FAK, clone 4.47Upstatemouse (mAb)WB, IP, IC, IH05-537
    FAK clone 2A7Upstatemouse (mAb)IP, IC05-182
    FAKUpstaterabbitWB, IP, IH06-543
    FAK, BC3UpstaterabbitIP, IC06-446
    FAKBioSourcerabbitIP, WBAHO0502
    FAKBD PharMingenrabbitWB, IP556368
    FAKBD Transductionmouse (mAb)WB, IP, IC, IH610087
    FAKCell SignalingrabbitWB, IH3285
    FAKBioSourcerabbitWB, IP, ICAH0502
    FAKBioSourcerabbitWB, IP, ICAMO0672
    FAKChemiconrabbitIPAB1605
    FAKChemiconmouse (mAb)WB, IP, ICMAB2156
    FAK (H-1)Santa Cruzmouse (mAb)WB, IP, IC, IHsc-1688
    FAK (A-17)Santa CruzrabbitWB, IP, ICsc-557
    FAK (C-20)Santa CruzrabbitWB, IP, ICsc-558
    FAK (C-903)Santa CruzrabbitWB, IP, IC, IHsc-932
    Pyk2UpstaterabbitWB, IP, IC06-559
    Pyk2, clone 74Upstatemouse (mAb)WB, IP05-488
    Pyk2UpstaterabbitWB, IP, IC07-437
    Pyk2Cell SignalingrabbitWB, IP3292
    Pyk2BD Transductionmouse (mAb)WB, IP, IC, IH610548
    Pyk2 (H-102)Santa Cruzmouse (mAb)WB, IP, ICsc-9019
    Pyk2 (N-19)Santa CruzrabbitWB, IP, ICsc-1514
    Pyk2 (C-19)Santa CruzrabbitWB, IP, ICsc-1515
    c-Src, clone GD11UpstatemouseWB, IP05-184
    c-Src, clone N6LUpstaterabbit (mAb)WB05-889
    c-Src, clone NL19Upstaterabbit (mAb)WB, IP05-772
    c-SrcBioSourcemouse (mAb)WBAHO1152
    c-SrcBioSourcerabbitWB44-655G
    c-SrcBioSourcerabbitWB44-656G
    c-SrcChemiconsheepWB, IPCB769
    c-Src, clone 36D10Cell Signalingrabbit (mAb)WB, IP, IC, IH2109
    c-Src, clone L4A1Cell Signalingmouse (mAb)WB, IP2110
    c-SrcCell SignalingrabbitWB, IP, IC, IH2108
    c-Src (H-12)Santa Cruzmouse (mAb)WB, IP, ICsc-5266
    c-Src (B-12)Santa Cruzmouse (mAb)WB, IP, ICsc-8056
    c-Src (N-16)Santa CruzrabbitWB, IP, ICsc-19
    c-Src (Src 2)Santa CruzrabbitWB, IP, ICsc-18
     aAbbreviation: mAb, monoclonal antibody; WB, western (immuno)blot; IP, immunoprecipitation; IC, immunocytochemistry; IH, immunohistochemistry.
     
    Table 14.7.2 Commercially Available Phospho-Specific Antibodies to FAK, Pyk2, and c-Srca
    AntibodyCompanySourceApplicationCatalog number
    FAK pY397BioSourcerabbitWB, IC, IH44-624G
    FAK pY397 clone 141-9BioSourcerabbit (mAb)WB, IC44-625G
    FAK pY407BioSourcerabbitWB, IC, IH44-650G
    FAK pY576BioSourcerabbitWB44-652G
    FAK pY577BioSourcerabbitWB, IC44-614G
    FAK pS722BioSourcerabbitWB44-588
    FAK pS732BioSourcerabbitWB44-590G
    FAK pS843BioSourcerabbitWB44-594
    FAK pY861BioSourcerabbitWB, IC44-626G
    FAK pS910BioSourcerabbitWB44-596
    FAK pY397 clone 14BD/Transductionmouse (mAb)WB, IC611722
    FAK pY397 clone 18BD/Transductionmouse (mAb)WB, IC611806
    FAK pY576/pY577Cell SignalingrabbitWB3281
    FAK pY397Santa CruzrabbitWB, ICsc-11765-R
    FAK pY397Santa CruzrabbitWB, ICsc-21868-R
    FAK pY407Santa CruzgoatWB, ICsc-16664
    FAK pY576Santa CruzrabbitWB, ICsc-16563-R
    FAK pY477Santa CruzgoatWB, ICsc-16665
    FAK pY576/pY577Santa CruzrabbitWB, ICsc-21831-R
    FAK pY576/pY577Santa CruzgoatWB, ICsc-21831
    FAK pS722Santa CruzgoatWB, ICsc-16662
    FAK pY861Santa CruzgoatWB, ICsc-16663
    FAK pS910Santa CruzgoatWB, ICsc-16666
    FAK pY925Santa CruzgoatWB, ICsc-11766
    FAK pY397Chemiconmouse (mAb)WB, ICMAB1144
    FAK pY397UpstaterabbitWB, IC07-012
    FAK pY576UpstaterabbitWB, IC07-157
    Pyk2 pY402BioSourcerabbitWB, IH44-618G
    Pyk2 pY579BioSourcerabbitWB, IC44-632G
    Pyk2 pY579/pY580BioSourcerabbitWB44-636G
    Pyk2 pY580BioSourcerabbitWB44-634G
    Pyk2 pY881BioSourcerabbitWB, IH44-620
    Pyk2 pY402Cell SignalingrabbitWB, IP3291
    Pyk2 pY402Santa CruzrabbitWB, ICsc-11767-R
    Pyk2 pY579Santa CruzgoatWB, ICsc-16822
    Pyk2 pY579/pY580Santa CruzgoatWB, ICsc-16824
    Pyk2 pY580Santa CruzgoatWB, ICsc-16823
    Pyk2 pY881Santa CruzgoatWB, ICsc-16825
    Pyk2 pY402 clone RR102Upstatemouse (mAb)IP05-679
    Active Src clone 28BioSourcemouse (mAb)WB, IC, IHAHO0051
    Src pY416BioSourcerabbitWB, IC44-660G
    Src pY527BioSourcerabbitWB, IH44-662G
    Src pY527 clone 31BD/Transductionmouse (mAb)WB612668
    Src pY416Cell SignalingrabbitWB, IC, IH2101
    Src pY416 clone 7G9Cell Signalingmouse (mAb)WB, IP2102
    Non-phospho Src pY527Cell SignalingrabbitWB2107
    Src pY527Cell SignalingrabbitWB, IH2105
    Src pY527Santa CruzgoatWB, ICsc-16846
    Src pY416 clone 2N8Upstaterabbit (mAb)WB05-857
    Src pY416 clone 9A6Upstatemouse (mAb)WB05-677
     aAbbreviations: mAb, monoclonal antibody; WB, western (immuno)blot; IP Immunoprecipitation; IC, immunocytochemistry; IH, immunohistochemistry.

Basic Protocol 2: In Vitro Kinase Assay

 Materials
  • Immunoprecipitated samples (Support Protocol 4, step )
  • FAK-Pyk2 kinase buffer (see recipe), 4°C
  • Src kinase buffer (see recipe), 4°C
  • 10 µCi/µl [32P]ATP (>3,000 Ci/mmol; PerkinElmer)
  • Magnesium/ATP cocktail (Upstate Biotechnology or see recipe)
  • 2× Laemmli SDS buffer (see recipe)
  • Coomassie blue stain (see recipe)
  • Molecular weight marker (Precision Plus, Bio-Rad)
  • Destaining solution (see recipe)
  • Microcentrifuge
  • 1.5-ml microcentrifuge tubes
  • 32°C water bath
  • Plexiglas shielding
  • Gloves
  • Geiger counter
  • Plexiglas box
  • Micro tube cap locks (RPI 145063; http://www.rpicorp.com)
  • Whatman 3MM filter paper
  • Gel dryer
  • Immobilon PVDF membrane (Millipore IPFL 000-10)
  • Additional reagents and equipment for SDS-PAGE (unit 6.1), autoradiography (unit 6.3), and transfer of proteins to membranes for immunoblotting (unit 6.2)

CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following guidelines provided by the local radiation safety officer (also see appendix 1A).

Alternate Protocol 1: FAK-PYK2 Poly Glu:Tyr Phosphorylation

 Additional Materials (also see Basic Protocol 2)
  • Immunoprecipitated samples (see Support Protocol 4, step )
  • 10 mg/ml poly(Glu:Tyr; 4:1) mol. wt. 20,000 to 50,000: sodium salt (Sigma-Aldrich) prepared in PBS and stored up to 2 years at –20°C in 1-ml aliquots
  • Magnesium/ATP cocktail (Upstate Biotechnology or see recipe)
  • 10 µCi/µl [32P]ATP (>3,000 Ci/mmol; PerkinElmer)
  • FAK-Pyk2 kinase buffer
  • 0.75% phosphoric acid
  • 100% acetone
  • Scintillation fluid (Sigma)
  • 2 × 2–cm Whatman 3MM filter paper squares
  • Conical 50-ml centrifuge tube
  • Scintillation vials
  • Scintillation counter
  • Additional reagents and equipment for preparing immunoprecipitated samples (Support Protocol 4)

CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following guidelines provided by the local radiation safety officer (also see appendix 1A).

Alternate Protocol 2: Measurements of Src-Associated Kinase Activity

 Additional Materials (also see Basic Protocol 2)
  • 10 mg/ml GST-FAK 853-1052 (Schlaepfer lab)
  • Enolase (Sigma-Aldrich E-0379), optional
  • 50 mM HCl, optional
  • 1 M PIPES, pH 7, optional
  • 30°C water bath, optional

CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following guidelines provided by the local radiation safety officer (also see appendix 1A).

Basic Protocol 3: Immunoblotting with FAK/PYK2 Phospho-Specific Antibodies

 Materials
  • 2× Laemmli SDS buffer (see recipe)
  • Immunoprecipitated samples (Support Protocol 4, step )
  • 100% and 20% methanol
  • Transfer buffer (see unit 6.2)
  • EZBlue gel staining reagent (Sigma)
  • BSA blocking buffer (see recipe)
  • Primary antibody (see Tables 14.7.1 and 14.7.2)
  • Tris-buffered saline with Tween (TBST, see recipe)
  • Secondary antibody: Horseradish peroxidase (HRP)-conjugated appropriate species (Pierce)
  • Enhanced chemiluminescence (ECL) western detection solution (Amersham RPN 2132)
  • Western blot stripping buffer (see recipe)
  • Immobilon PVDF membrane (Millipore)
  • Rotating shaker
  • Additional reagents and equipment for gel electrophoresis (unit 6.1) and electrophoretic transfer (unit 6.2)

Basic Protocol 4: Haptotaxis Motility Assay: Matrix-Stimulated Migration

 Materials
  • FAK–/– and FAK+/+ cells, subconfluent (see Support Protocol 1)
  • Human plasma fibronectin (Sigma-Aldrich F2006): 2 to 10 µg/ml in replating and migration medium (see recipe), 37°C
  • BSA-coating control: 10 µg BSA/ml in replating and migration medium (see recipe)
  • Trypsin/EDTA: 0.25% (w/v) trypsin/1 mM EDTA (Invitrogen)
  • Trypsin inhibitor solution (see recipe)
  • PBS++: phosphate-buffered saline (PBS; appendix 2A) with 0.1 g/liter CaCl2 and 0.5 mM MgCl2
  • Cell fixative: PBS with 1.85% (v/v) formaldehyde and 0.05% (v/v) glutaraldehyde
  • 10% (w/v) crystal violet stain (Sigma) in ethanol
  • 0.1 M sodium borate, pH 9.0
  • Parafilm
  • Millicell PCF chamber inserts, 8-µm pore (Millipore PITP01250)
  • Flat-tipped forceps
  • 15-ml centrifuge tube
  • Tabletop centrifuge
  • Hemacytometer
  • 24 well-tissue culture dishes (Costar)
  • Cotton swabs
  • Inverted light microscope
  • Spectrophotometer with A600 capability, optional
  • Additional reagents and equipment for serum-starving cells (Support Protocol 2) and counting cells (unit 1.1)

Alternate Protocol 3: Analyzing Cell Motility Using Plasmid-Transfected Cells

 Additional Materials (also see Basic Protocol 4)
  • pcDNA3.1 FAK (contact Schlaepfer lab)
  • FAK–/– cells (see Support Protocol 1)
  • pcDNA3.1 LacZ (Invitrogen)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • PBS++: phosphate-buffered saline (appendix 2A) with 0.1 g/liter CaCl2 and 0.5 mM MgCl2
  • Opti-MEM I reduced-serum medium (Invitrogen)
  • PLUS reagent (Invitrogen)
  • Lipofectamine (Invitrogen)
  • Cell growth medium (see recipe) containing 20% (v/v) FBS (instead of 10%)
  • Starvation medium: Cell growth medium (see recipe) with FBS reduced to 0.5%
  • Lac Z staining solution (see recipe)
  • Additional reagents and equipment for bacterial transformation (Seidman et al., 1997), plasmid miniprep (Engebrecht et al., 1991), and DNA quantification (appendix 3D)

Basic Protocol 5: Scratch-Wound Healing Assay With Time-Lapse Imaging

 Materials
  • Extracellular matrix molecule (ECM) of interest (e.g., 2 µg fibronectin/ml PBS)
  • 70% confluent 24-hr serum-starved cells (see Support Protocol 2)
  • DMEM with and without 0.5 µg/ml mitomycin-C
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Serum, optional
  • Mitomycin-C (Sigma)
  • Medium 199 (Invitrogen) with 0.5 µg/ml mitomycin-C
  • Mineral oil (Sigma)
  • 25-mm glass coverslips (1 oz., Fisher) and 6-well tissue culture plates or
  • 35-mm Bioptechs delta-T dishes (Fisher)
  • Forceps
  • 1- to 10-µl micropipet tips
  • Transfer pipets, sterile
  • Inverted microscope with 20× objective, 37°C heated stage, and acquisition/analysis software (e.g., Improvision Openlab; https://www.improvision.com)
  • Etched-grid coverslips (Bellco), optional

Basic Protocol 6: Immunolocalization of Focal Adhesion Proteins

 Materials
  • Matrix-coating substrates: prepared according to manufacturer's directions and diluted (commonly to 10 µg/ml) in PBS (appendix 2A)
    • Fibronectin, human plasma (Roche)
    • Laminin, human placenta (Sigma)
    • Vitronectin, human plama (Sigma)
    • Poly-l-lysine (70,000–150,000 or 150,000–300,000; Sigma)
    • Collagen, Type I, human placenta (Calbiochem)
    • Collagen, Type II, bovine (Calbiochem)
    • Collagen, Type IV, human placenta (Calbiochem)
    • Collagen, Type V, human (Calbiochem)
  • FAK–/– and FAK+/+ cells (see Support Protocol 1)
  • Replating and migration medium (see recipe), warm or growth medium (see recipe), 37°C
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 3.8% (w/v) paraformaldehyde fixative (see recipe)
  • Acetone, cold (stored at –20°C) or
  • 0.5% (v/v) Triton X-100/0.05% (v/v) Tween 20/PBS or
  • 0.2% Triton X-100/PBS or
  • 0.2% (v/v) Triton X-100/3.8% (w/v) paraformaldehyde/PBS or
  • 0.1% (v/v) Triton X-100/0.1% (w/v) sodium citrate or
  • Methanol, cold
  • Blocking antibody (e.g., ChromPure donkey IgG, unconjugated; Jackson Immunoresearch) or
  • 2% (w/v) BSA in PBS
  • 1 to 10 µg/ml PBS (appendix 2A) primary antibodies for focal adhesion markers
    • anti-paxillin (ZO35; Zymed/Invitrogen)
    • anti-vinculin (VIN-11-5; Sigma)
    • anti-FAK (clone #77; BD-Transduction)
    • anti-FAK (Ab-1; LabVision)
    • anti-phospho Y397FAK (BioSource)
    • anti-active Src family members (clone #28; BioSource)
  • Secondary antibodies: e.g., fluorescein (FITC)-conjugated donkey antibodies (excitation/emission maxima 492/520 nm; Jackson Immunoresearch) or FITC-conjugate
    • anti-mouse IgG
    • anti-mouse IgM
    • anti-rabbit IgG
    • anti-goat IgG
    • anti-rat IgG
  • Rhodamine X (RRX)-conjugated donkey antibodies (excitation/emission maxima 570/590 nm; Jackson Immunoresearch) or Rhodamine X (RRX)-conjugated
    • anti-mouse IgG
    • anti-mouse IgM
    • anti-rabbit IgG
    • anti-goat IgG
    • anti-rat IgG
  • Hoechst 33342 (excitation/emission maxima 350/460; Molecular Probes)
  • Vectashield mounting medium (Vector)
  • Nail polish, clear
  • 12-mm round coverslips, German glass (Bellco Glass)
  • 4-well tissue culture plates (Nunc)
  • 12-well tissue culture plates (Costar)
  • 18- to 22-G needle with a slightly bent tip
  • Flat-ended forceps with beveled, unserrated tips, stainless steel (Millipore)
  • 6-well tissue culture plates
  • Rotating platform shaker
  • Vacuum source
  • Porcelain spot plates with 12 cavities (CoorsTek)
  • Light microscope with fluorescence excitation and detecting capability

Alternate Protocol 4: Immunolocalization of Actin Stress Fibers

 Additional Materials (also see Basic Protocol 6)
  • 0.4 to 1 U/ml (10 to 30 nM) fluorescein-conjugated phalloidin (Molecular Probes) or rhodamine-conjugated phalloidin (Molecular Probes) in PBS
  • Rotating platform shaker
  • Vacuum source
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Figures

  • Figure 14.7.1
    Measurements of Pyk2 of FAK-associated in vitro kinase activity. Lysates from serum-starved (attached), suspended (S) fibronectin-plated (FN), and poly-l-lysine-plated (PL) FAK–/– cells (A and B) or FAK+/+ cells (C and D) were prepared and divided into equal aliquots for either Pyk2 immunoprecipitates (IPs) or FAK IPs, respectively. (A) Pyk2 IPs were labeled by the addition of [-32P]ATP in an in vitro kinase (IVK) assay. 32P-labeled proteins were transferred to membranes and visualized by autoradiography. (B) The same membrane shown in (A) was cut and analyzed by either anti-Pyk2 or anti-Src family PTK blotting. (C) FAK IPs were labeled by the addition of [-32P]ATP in an IVK assay and the same membrane (D) was cut and analyzed by either anti-FAK or anti-Src family protein-tyrosine kinase (PTK) immunoblotting. Molecular weight standards are indicated in kDa to the left. (previously published in the EMBO Journal; Sieg et al., 1998)

    View Image

Literature Cited

 Internet Resources
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