Total cell protein extracts were prepared using RIPA buffer as described previously [12 (link)]. Membranes were blocked for 1 h with 10% non-fat milk or 5% BSA in TBS containing 0.1% Tween-20, and then incubated overnight at 4°C with the primary antibody at the dilutions recommended by the manufacturer.
The primary antibodies against FAK and phospho-FAK (Tyr397) were purchased from Cell Signaling (Danvers, MA, USA), and the anti-β-actin from Sigma-Aldrich (Steinheim, Germany). HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody. Blots were developed using Lumi-Light Plus Reagent (Roche, Barcelona, Spain), and the autoradiograms were scanned using a GS-800 calibrated densitometer and analyzed using Quantity One software (Biorad, Berkeley, CA,USA).
The primary antibodies against FAK and phospho-FAK (Tyr397) were purchased from Cell Signaling (Danvers, MA, USA), and the anti-β-actin from Sigma-Aldrich (Steinheim, Germany). HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as the secondary antibody. Blots were developed using Lumi-Light Plus Reagent (Roche, Barcelona, Spain), and the autoradiograms were scanned using a GS-800 calibrated densitometer and analyzed using Quantity One software (Biorad, Berkeley, CA,USA).
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