Cd151

Cells were harvested, washed twice with PBS, and lysed with RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Shanghai, China) with protease inhibitors (Thermo Fisher Scientific). Tumor tissues retrieved from −80°C storage were rapidly immersed in liquid nitrogen followed by lysis with RIPA Lysis Buffer and protease inhibitors. Protein concentrations were determined with a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Total protein (50 μg/lane) was separated by 10% SDS-PAGE and was transferred to nitrocellulose membranes. After blocking for 60 min with 5% milk solutions prepared in PBS, the membranes were incubated overnight at 4°C with 1:1000 dilutions of the following primary antibodies: CD151 (#5901), FAK (#2146-1), and CD29 (#1798-1; all from abcam, Cambridge, UK), Akt (#4685), p-Akt (#4060), p38 (#8690), p-p38 (#4511), mTOR (#5536), p70S6 (#9208), p65 (#8242), and p-p65 (#3033; all from Cell Signaling Technology, Danvers, MA, USA). After the membranes were washed, they were incubated for 1 h with the appropriate peroxidase-conjugated secondary antibody (1:1000 dilution). Membranes were developed using the Odyssey Two-Color Infrared Laser Imaging System (LI-COR Biosciences, Lincoln, NB, USA). The signal generated by actin (#4973; Cell Signaling Technology) was used as an internal control.