2.8. Western Blot Analysis

The HepG2 cell line was harvested and rinsed using PBS. According to the manufacturer's instructions, the RIPA lysis buffer supplemented with the complete protease inhibitor (Roche, Switzerland) was used to lyse HepG2 cells. 25 µg of proteins was incubated and then was isolated onto 12% SDS-PAGE followed by transfer onto the PVDF membranes. Thereafter, the 3% skimmed dry milk powder was used to block PVDF membranes followed by incubation with the primary antibody at 4°C for 12 h according to the specifications [29]. Later, secondary IgG antibody (goat anti-mouse or anti-rabbit IgG) was used to incubate the membranes for another 2 h. After washing, the ECL kit (Amersham Pharmacia Biotech) was used to react with membranes. In the study, the following antibodies (San Diego, CA, USA) were utilized: PARP, cleaved PARP, Bax, Bcl-2, cytochrome c, AIF (all dilutions, 1 : 800), together with beta-actin (1 : 2000).