Ap14272b

Frozen liver tissues or cells were treated with protein extraction reagent supplemented with protease inhibitor cocktail (Bimake, B14002). Extracted protein samples were used for electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membrane. The membranes were blocked with 5%(w/v) skim milk (BD Bioscience, USA) for 1 h at room temperature, and then incubated with the following primary antibodies overnight at 4 °C: TWEAK (Abcam, ab37170, 1:1000), Tnfrsf12a(Abcam, ab109365, 1:1000), Phospho-RIPK1 (Cell Signaling Technology, 31122S, 1:1000), cleaved caspase-8 (Cell Signaling Technology, 8592T, 1:1000), cleaved caspase-3 (Cell Signaling Technology, 9664S, 1:1000), cleaved PARP (Cell Signaling Technology, 94885S,1:1000), RIPK3 (Cell Signaling Technology, 15828, 1:1000), MLKL (Abgent, AP14272B, 1:1000), GSDMD (Abcam, ab219800, 1:1000), GSDME (Abcam, ab215191, 1:1000), Tubulin (Sigma, T6199, 1:1000), GAPDH (Proteintech, 10494-1-AP, 1:5000). Original western blots are presented in Supplementary file.

Liver tissues or cells were lysed and homogenized in RIPA buffer supplemented with a protease inhibitor cocktail (Roche). Total protein was quantified using the BCA Protein Assay Kit (Thermo Fisher). Then protein extracts were boiled at 100 °C for 10 min in sample buffer (50 mM Tris-HCl, 2% w/v SDS, 10% glycerol, 1% β-mercaptoethanol, 0.01% bromophenyl blue (pH 6.8)). Cell lysates were separated on SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were first incubated with blocking buffer (TBS with 0.05% Tween 20, 10% nonfat milk) for 1 h at room temperature and then incubated overnight at 4 °C in buffer containing primary antibody. After a thorough wash, cells were incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000) for 1 h. Immunostaining was visualized using Amersham ECL Plus Western Blotting detection reagents (GE Healthcare) and ChemiDoc imaging system (Bio-Rad).
Antibodies used include human MLKL (1:1000; Abcam ab194699), human p-MLKL (1:1000; CST 91689S), mouse MLKL (1:1000; Abgent AP14272B), mouse p-MLKL (1:1000; Abcam ab196436), RIP1 (1:1000; CST 3493), p-RIP1 (1:1000; CST 31122), RIP3 (1:1000; Abcam ab152130), p-RIP3 (1:1000; CST 91702), α-SMA (1:1000; Invitrogen MA1-06110), Vimentin (1:1000; Abcam ab8978), Smad2/3 (1:1000; CST 8685), p-Smad2/3 (1:1000; CST 8828), Gapdh (1:10,000; CST 8884).