Antibodies against cytochrome c

For Western blotting analyses, the collected cells were rinsed in PBS and lysed in NETN lysis buffer (Bethyl Laboratories, Inc., Montgomery, TX, USA). Equal amounts of protein were separated on 10%–12% SDS-polyacrylamide gels, transferred to PVDF membranes, and blocked with 5% nonfat dry milk in TBST for 1 h. The membranes were then incubated with primary antibodies overnight at 4 °C. Antibodies against PARP, E-cadherin, N-cadherin, vimentin, cyclin A2, cyclin D1, CDK2, and β-actin were purchased from Cell Signaling Technology (Beverly, MA, USA) (all used at 1:1000 dilutions). Antibodies against cytochrome c were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) (1:2000 dilutions). After being washed 3 times with TBST for 10 min, the membranes were incubated with appropriate secondary antibodies in TBST for 1 h. After several washes of TBST, the blots were developed by enhanced chemiluminescence (ECL) solution (Bio-Rad, Hercules, CA, USA); Western blots were quantified using ImageJ software (1.8.0_112, Windows).

Antibodies against cytochrome c, poly (ADP-ribose) polymerase (PARP), and β-actin were purchased from Santa Cruz Biotechnology. Antibodies against caspase-8, caspase-9, Fas, TNFR1, TNF-α, DR4, DR5, and p53 were purchased from Abcam (Hong Kong). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Pierce Biotechnology. Equal amounts of cell lysates were separated by SDS-PAGE and electrotransferred onto immobilon transfer membranes (Millipore, Bedford, MA). The membranes were blocked with 5% skim milk and probed with the indicated antibodies. The blots were washed and incubated with an HRP-coupled anti-mouse or anti-rabbit IgG antibody, followed by detection with ECL-enhanced chemiluminescence detection reagents (Amersham Biosciences, Piscataway, NJ). β-actin, α-tubulin or COXII were used as loading controls.