Survivin antibody

Cells ($300,000) were plated in 60-mm diameter plates and allowed to grow for 48 hours. The old medium was then replaced with 2 mL fresh RPMI medium and then the cells were treated with inhibitors. After treatment, cells were harvested, washed, and lysed in lysis buffer (50 mmol/L HEPES buffer, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L orthovanadate, 10 mmol/L sodium pyrophosphate, 10 mmol/L sodium fluoride, 1% NP-40, and a cocktail of protease inhibitors). Proteins were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked with 5% nonfat-milk solution and blotted with appropriate primary antibody followed by peroxidase-labeled secondary antibody. Bands were visualized by enhanced chemiluminescence detection kit from Pierce Biotech (Rockford, IL). To be accepted as valid, protein blots were analyzed at least in two independent experiments showing similar results. Antibodies against TRIB2, JNK (p), pAKT, FOXO3, and pH2A.X were from Cell Signaling Technology. Survivin antibody was purchased from R and D Systems (Minneapolis, MN), and antibodies against, Bcl-xL, cyclin D1, ATF3, caspase-3, and GAPDH were from Santa Cruz Biotechnology (Santa Cruz, CA). Ki-67 antibody was purchased from Sigma Chemical CO (St. Louis, MO).