Western blotting

Protein extracts from cultured cells were prepared using an extraction buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl, 12.5 mM β-glycerophosphate, 1.5 mM MgCl₂, 2 mM EGTA, 10 mM NaF, 2 mM DTT, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 20 µM aprotinin, and 0.5% Triton X-100. The extracts were boiled in SDS sample buffer containing 2-mercaptoethanol at 95°C for 5 min, resolved on SDS-PAGE, and transferred onto Hypond-P membranes (GE Healthcare). The membranes were immunoblotted with the indicated antibodies, and the bound antibodies were visualized with horseradish peroxidase–conjugated antibodies against rabbit or mouse IgG using the ECL Western blotting system (GE Healthcare).

For testing oligomerization of endogenous MLKL, protein extracts from cultured cells were boiled in 2-mercaptoethanol-free SDS sample buffer at 85°C for 10 min, and then resolved on SDS-PAGE.