Anti rip3

Protein isolation and Western Blot were conducted as previously described^{26 ,27 (link)}. In brief, GCs were lysed after 1 to 5 days of culture using RIPA buffer containing protease and phosphatase inhibitors (PI, Thermo Fisher Scientific, Waltham, USA). A total of 10 µg protein per lane was loaded on a 12 % SDS gel and run under constant current (30 mA/gel). After blotting (100 V, 65 min) and blocking with 5 % non-fat dry milk in Tris-buffered saline with Tween 20 (TBS-T, 50 mM Tris-HCl, 150 mM NaCl, 0.1 % Tween 20, pH 7.4), anti-pRIP1(S166), anti-pRIP3(S227) both from Cell Signaling Technology (Danvers, MA, USA) and anti-pMLKL(T357/S358) antibodies were administered to decorate these phosphorylated proteins. To visualize specific binding, HRP-conjugated goat anti-rabbit antibody (Jackson ImmunoResearch Inc., West Grove, PA, USA) was used. As loading controls, anti-RIP1, anti-RIP3 (both from Cell Signaling Technology, Danvers, MA, USA), anti-MLKL (ab184718, Abcam, Cambridge, UK) and anti-β-actin (A5441, Sigma-Aldrich, St. Louis, MI, USA) antibodies were used. Preabsorption of anti-pMLKL was previously published^{27 (link)}. All experiments were carried out five times if not described otherwise.

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.