Western Blotting Assay Protocol

Western blotting assays were carried out as previously described^{29 (link),30 (link)}. Briefly, tissue samples or cell lysates were prepared in ice-cold radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Nantong, Jiangsu, China). Equal amounts of total protein were subjected to SDS-PAGE and transferred onto PVDF membranes, which were blocked and incubated overnight with the following primary antibodies: anti-RIP3 monoclonal antibody (#95702, Cell Signaling Technologies) or anti-RIP3 polyclonal antibody (ab152130, Abcam, Cambridge, MA, USA), anti-phospho-RIP3 monoclonal antibody (ab205421, Abcam, Cambridge, MA, USA), anti-MLKL polyclonal antibody (#28640, Cell Signaling Technologies) or anti-MLKL monoclonal antibody (#14993, Cell Signaling Technologies), anti-phospho-MLKL monoclonal antibody (ab208910, Abcam, Cambridge, MA, USA) or anti-phospho-MLKL monoclonal antibody (#91689, Cell Signaling Technologies), or anti-β-actin monoclonal antibody (sc-47778, Santa Cruz Biotechnology, CA, USA). After washing, the membranes were incubated with the appropriate secondary antibodies conjugated with horseradish peroxidase (HRP). Immune-reactive signals were visualized by an enhanced chemiluminescence (ECL) kit (Millipore, USA) on a Syngene PXi6 Access imaging system (Frederick, MD). The band intensities were quantified using Image-Pro Plus 6.0.