Western Blotting Analysis of Apoptosis-Related Proteins

To evaluate the expression of apoptotic proteins, PC3 cells were subjected to western blotting analysis after treatment with EGCG-loaded NPs. For this purpose, cells were lysed using freeze/thaw cycles, and the cell lysates were centrifuged at 6,000 g at 4°C. Equal amounts of cell protein lysates were electrophoretically separated through sodium dodecyl sulfate (SDS) polyacrylamide gel, and the separated proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes, which were putted into PBS containing 5% (w/v) nonfat dry milk for 1 h and then probed with primary antibodies [i.e., rabbit poly-clonal anti-caspase-9 and anti-poly (ADP-ribose) polymerase (PARP)]; mouse monoclonal anti-caspase-8, anti-caspase-3 and anti-β-actin) overnight at 4°C. Membranes were incubated with horseradish peroxidase-conjugated secondary anti-bodies for 1 h, and immune complexes were detected enhanced chemiluminescence (Amersham Life Science, United States).