Western Blot Analysis of Apoptosis Markers

Cells were lysed using RIPA buffer supplemented with aprotinin (Sigma), and Halt Protease Inhibitor Cocktail (100X) (Thermo Scientific). Protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Bio-Rad, Mississauga, ON). Primary antibodies used in this study include caspase-1 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-1 (AdipoGen Life Science, San Diego, CA), caspase-3 (Sant Cruz Biotechnology, Dallas, TX), cleaved caspase-3 (Cell signaling technology, Danvers, MA), HMGB1 (Cell signaling technology, Danvers, MA), MLKL (Abcam, Boston, MA), phosphorylated MLKL (Abcam, Boston, MA), CRT (Abcam, Boston, MA), and GAPDH (Sant Cruz Biotechnology, Dallas, TX). The expression levels Relative expression of proteins were visualized using the corresponding secondary HRP-conjugated antibodies and Amersham ECL select western blotting detection reagent, as described previously (46 (link)).