Anti pfak tyr397

Protein extracts (cleared lysates) were obtained from cells grown to 70% confluence using standard RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, pH 7.4) containing protease inhibitor cocktail (Quartett GmbH, Berlin, Germany) and 1 mM sodium orthovanadate (Na₃V0₄; Sigma). Cells were collected using a cell scraper, incubated on ice for 5-10 min and centrifuged at 12,000 x g for 20 min at 4°C. Proteins (40 μg) were separated by 10% SDS-PAGE, transferred onto nitrocellulose membranes [7 (link)], blocked with 5% BSA in TBS-Tween for 1 h and incubated overnight at 4°C with the following primary antibodies diluted in 2% BSA: rabbit polyclonal anti-Calretinin (1:10,000; CR 7699/4), rabbit polyclonal anti-FAK (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-p-FAK Tyr³⁹⁷ (1:1,000; Cell Signaling Technology), and rabbit polyclonal anti-GAPDH (1:5,000; Sigma); followed by incubation with secondary goat anti-rabbit or anti-mouse (HRP)-labeled antibodies (Sigma) at a dilution of 1:10,000. The signals were detected as described in [7 (link)]. For the EMT analysis membranes were incubated overnight at 4°C with a rabbit monoclonal anti-E-cadherin antibody (1:1,000; #3195; Cell Signaling Technology) and a mouse monoclonal N-cadherin (1:1,000; # 610920; BD Bioscience).