Western Blot Analysis of Apoptosis Markers

Cells were harvested and lysed as previously described [67 (link)]. Cell lysates were separated by 7.5% or 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). To detect the protein of interest, anti-PARP (1:3000), anti-caspase-3 (1:1000), anti-cleaved caspase-3 (1:1000), anti-histone H3 (1:3000), anti-Phospho-RIP1 (S166) (1:1000), anti-RIP3 (1:1000), anti-β-actin (1:3000) (all from Cell Signaling Technology, Danvers, MA, USA), Phospho-RIP3 (Abcam, Cambridge, UK), and RIP1 (BD Biosciences) were used as primary antibodies. HRP-conjugated goat anti-mouse and anti-rabbit IgG (Santa Cruz Biotechnology, Dallas, TX, USA) were used as secondary antibodies.