Cellular Protein Analysis by Western Blotting

Cellular protein was analyzed by Western blotting as reported [21] (link). Briefly, cellular protein was extracted with detergent, and the protein concentration of the lysates was determined for each sample with the BCA Protein Assay Kit (Beyotime, China). Equal amounts of protein were separated by 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Pierce Biotechnology, Inc. Rockford, IL, USA). After blocking with 5% skim milk, the PVDF membranes were incubated with p53, p21, cyclin E1, Fas-L, cleaved caspase 3,8,9 or β-actin primary antibody (all rabbit IgGs; 1∶1000; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. The membranes were washed three times in PBST (10 mM NaH₂PO₄, 130 mM NaCl, 0.05% Tween 20) and then probed with a horseradish peroxidase-conjugated secondary antibody (goat anti rabbit IgG; 1∶5000; Molecular Probes, Invitrogen) for 1 h. Protein expression was visualized by the enhanced chemiluminescence (ECL) western blotting detection reagent (GE Health Care Bio-Sciences, Piscataway, NJ, USA).