Phosphorylated p p53

Whole cell extracts were lysed in cell lysis buffer (Biosesang, Inc., Seongnam, Korea). Equal quantities of protein (30 µg) were separated on 8–12% SDS-PAGE gels and were subsequently transferred onto a polyvinylidene difluoride membrane (GE Healthcare Life Sciences, Freiburg im Breisgau, Germany). After blocking the membranes with 1% bovine serum albumin and 2% skimmed milk for 1 h, the membranes were incubated at 4°C overnight with the appropriate primary antibody, and were washed three times in phosphate buffered saline with 0.01% Tween-20. The membranes were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibodies. In order to visualize the protein bands, the membranes were treated with enhanced chemiluminescence kit solution (DoGen) and exposed to X-ray film (AGFA Healthcare, Mortsel, Belgium). Anti-PARP, caspase-3, caspase-9, cyclin-dependent kinase (CDK)4, phosphorylated (p-)p53 and p-murine double minute 2 (MDM2) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-CDK1, CDK2, cyclin E, cyclin A, cyclin B, p21, p53 and Bcl-2 antibodies were purchased from Santa Cruz Biotechnology, Inc. Anti-cyclin D and Bcl-xL antibodies were obtained from BD Biosciences (San Jose, CA, USA). The anti-tubulin antibody was obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA).