4.7.2. Western Blotting

To confirm the data of quantitative PCR, we evaluated gene expressions in protein level. Briefly, total protein was extracted from cells before and after treatment. Equal amounts of extracted proteins (40 mg/sample) were separated by SDS polyacrylamide gel electrophoresis and transferred to 0.45-mm polyvinylidene difluoride membrane. The membrane was blocked with 5% (*w/v*) non-fat dried milk (Difco/Becton Dickinson, Franklin Lakes, NJ, USA) and incubated for 2 h at room temperature with primary antibody [1:200, BCL-2 (sc-492), BAX (sc-7480), and β-actin (sc-47778)]. Mouse anti-rabbit IgG-HRP in 5% defatted dry milk-TBS-0.1% Tween (Santa Cruz Biotechnology, Dallas, TX, United States) were employed to visualize proteins. All steps were performed at room temperature. All

signals were visualized using enhanced Western Blotting Luminol Reagent (Santa Cruz Inc., Dallas, TX, USA).
