Anti bcl2 and anti bax

Tissue samples and cells were lysed in ice-cold radioimmunoprecipitation assay buffer buffer (Beyotime, Shanghai, China) with fresh-added protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). Total cell lysates with equal amount of protein for each sample were loaded to sodium dodecyl sulfate-polyacrylamide gel and then transferred to nitrocellulose membranes (Millipore, Bradford, MA, USA). The membranes were incubated with 5% non-fat milk in Tris buffer containing Tween-20 (TBST) for 1 h at room temperature to block non-specific binding. Subsequently, the membranes were probed with primary antibodies (anti-STK39 and anti-c-Myc [Abcam, Cambridge, MA, USA]; anti-Bcl-2 and anti-Bax [Santa Cruz Biotech, Santa Cruz, CA, USA]; anti-p53, anti-p-p38, anti-p38, and anti-GAPDH [Cell Signaling Technology, Danvers, MA, USA]) following the manufacturers’ instructions. After washing three times with TBST, the corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime) were added. After incubation for 1 h at room temperature and after washing three times with TBST, signals were detected with enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA). The band density was quantified using ImageJ (http://rsb.info. nih.gov/ij/, Bethesda, MD, USA).