Western Blot Analysis of Apoptotic Markers

After sample treatments, MCF-7 cells incubated in 6-well plates were collected and lysed with RIPA buffer containing 1 mM phenylmethylsulfonyl fluoride on ice. The amounts of each protein were determined using the Pierce™ BCA Protein Assay Kit. Equal amounts of proteins (20 μg/lane) were separated by electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred onto polyvinylidene difluoride membranes [37 (link)]. After blocking with 5% skim milk for 1 h, the proteins in the membrane were incubated at 25 °C for 1 h with primary antibodies against BID, Bax, Bcl-2, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, PARP, and GAPDH. Following incubation with HRP-conjugated anti-rabbit secondary antibodies at room temperature for 1 h, the expressed proteins were reacted using ECL Advance Western blotting detection reagents and visualized with a FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany) according to the manufacturer’s instructions.