Total proteins were extracted with a RIPA buffer (Sigma Chemical Co., St. Louis, MO, USA). The mitochondria and cytosol fractions were isolated using a Mitochondria Isolation Kit (Abcam, Cambridge, MA, USA) following the manufacturer’s procedure. Western blotting was conducted as describe in our previous report [24 (link)]. Briefly, 25 μg of total extracted proteins were separated via 10–12% SDS-PAGE prior to nitrocellulose membranes transfer by electroblotting, which was then blocked with 5% skimmed milk for 1 h. Blots were then incubated with specific antibodies HO-1 (1:200), Bax (1:200), Bcl-2 (1:200), cytochrom C (1:200), β-actin (1:500), and Cox IV (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and caspase-3 (1:500) (Cell Signaling Technology, Beverly, MA, USA) overnight at 4 °C;. The horseradish peroxidase-conjugated antibodies were regarded as the secondary antibody. The bands were detected by chemiluminescence using the ECL Western blotting assay kit (Life Technologies, Seoul, Korea) and visualized by Davinch-Chemi Imager™ (CAS-400SM, Core Bio, Seoul, Korea).
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