Protein a g sepharose beads

R-H460 cells were transfected with vectors under the indicated experimental conditions for 48 h. The cells were washed twice with PBS, harvested, and lysed for 30 min in NP-40 buffer [50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% NP-40, and 100X protease and phosphatase inhibitor cocktail]. Samples were diluted to 500 µg of protein in 800 µl of buffer and pre-cleared for 1 h at 4 °C with 50 µl of a 50% slurry of protein A/G-Sepharose beads (GE Healthcare). After brief centrifugation to remove pre-cleared beads, 1 µg of antibody against HA (Santa Cruz Biotechnology) was added to each sample and incubated on a rocking platform at 4 °C overnight. The immune complex was precipitated by incubation with 40 µl of protein A/G-Sepharose beads at 4 °C for 4 h. The beads were washed thrice with immunoprecipitation buffer and then boiled with sample buffer [0.1 M Tris-HCl (pH 6.8), 4% SDS, 40 mM EDTA, 20% glycerol, and β-mercaptoethanol].

Immunoprecipitation and kinase reactions were performed as previously described [32 (link)]. Briefly, equal amounts of total cell lysate (600 μg) were immunoprecipitated for FAK with anti-FAK antibody (BD Bioscience, Mississauga, ON) and 20 μl of Protein A/G sepharose beads (GE Healthcare, Uppala, Sweden) while rotating for 2 hours at 4°C. The beads were then washed 3 times with 200 mM NETN (20 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 200 mM NaCl, 0.5% Igepal CA-630) followed by one wash in kinase buffer (0.25 mM NaVO₃, 20 mM Tris-HCl, pH 7.4, 1 mM NaF, 10 mM β-glycerophosphate, 1 mM dithiothreitol, 15 mM MgCl₂). DMSO (vehicle control) or FAK inhibitor PF-228 at 1 or 5 μM concentrations were then added to the reaction in 20 μl of kinase buffer and incubated for 20 minutes at 30°C. The kinase assay was then initiated with 1 μl of [³²P] γATP (5 μCi/μl) and incubated for 30 minutes at 30°C with mixing every 10 minutes. The reaction was terminated following the addition of 4X SDS sample buffer. The samples were resolved on a 7.5% polyacrylamide gel and transferred to PVDF membrane. The membrane was subjected to autoradiography for development of FAK phosphorylation events and probed for total FAK levels by western blotting as described below.

Transfection was performed using Lipofectamine 2000 (Invitrogen). Cells were harvested 24–48 h after transfection and lysated in EBC lysis buffer (0.5% NP-40, β-mercaptoethanol supplemented 50 mM Tris, pH 7.6, 120 mM NaCl, 1 mM EDTA, 50 mM NaF) and with protease inhibitor cocktail (Roche). For immunoprecipitation, 800 mg lysates were incubated with the appropriate antibody for 3 h at 4 °C followed by incubation for 1 h with Protein A/G sepharose beads (GE Healthcare). The resulting immunoprecipitates were washed at least three times in NETN lysis buffer (150 mM NaCl, 1 mM EDTA, 50 mM Tris–HCl, pH 7.8, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 0.5 μg ml⁻¹ leupeptin and 0.5 μg ml⁻¹ pepstin) before being resolved by SDS-PAGE and immunoblotted with the indicated antibodies.