Caspase-3 Cleavage Analysis by Western Blotting

Cells were scraped and lysed in buffer containing 10 mM Tris HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM ethylene glycol tetraacetic acid (EGTA). The cell lysates were then centrifuged at 14,000 g for 30 min. Samples were assayed in a spectrophotometer for protein concentration using the Bradford assay (Bradford, 1976) to normalize cell extracts to equal protein concentration. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes. Membranes were blocked overnight in PBS containing 0.1% Tween-20 and 5% non-fat dry milk. Blots were incubated with cleaved caspase-3 antibody (1:1000) and with HRP-linked secondary antibody (1:1000) for 1 h. Blots were then visualized with enhanced chemiluminescence system.