Fluorchem m system

SCs were washed twice with cold PBS and incubated in a lysis buffer (RIPA, BioTeke, Beijing, China) containing 1 mmol/L protease inhibitor PMSF (Sigma, St. Louis, MO, USA) and phosphatase inhibitors (Invitrogen, Carlsbad, CA, USA) on ice, followed by centrifugation at 12,000 g and 4°C for 15 min. Protein concentration was determined using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, USA). Protein samples (15 μg) were loaded onto a 10% polyacrylamide gel and transferred onto PVDF membranes using semidry methodology, as previously described [36 (link)]. Membranes were probed using specific antibodies: anti-FAK (#3285), anti-phospho-FAK (Tyr397; #8556), anti-paxillin (#3283), anti-phospho-paxillin (Tyr118; #2541), anti-Akt (#9272) or anti-phospho-Akt (Ser473; #9271), which were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin (AP0060), anti-rabbit IgG (BS13278) and anti-mouse IgG (BS30503) were obtained from Bioworld Technology (Louis Park, MN, USA). The proteins were visualized with the ECL-Plus Western blotting reagent in a FluorChem M system (Cell BioSciences, San Leandro, CA, USA). Band density was analyzed by Image J (http://rsb.info.nih.gov/ij/).

Cell lysates were subjected to 12% SDS-PAGE and bound to Hybond-C Extra nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ) using a semi-dry blotting system (Amersham Biosciences). After blotting, membranes were blocked and incubated with mouse primary antibodies followed by horseradish-peroxidase (HRP)-conjugated anti-mouse IgG (KPL). Bands were visualized using SuperSignal^® West Femto Trial Kit (Thermo Scientific/Pierce, Rockford, IL Rockford, IL) as an enhanced chemiluminescence substrate for HRP and then scanned using the FluorChem M system (Cell Biosciences, Santa Clara, CA).