Fig. 1. Endogenous MLKL is ubiquitylated during necroptosis.

a Quantification of propidium iodide positive (PI⁺) HT-29 cells upon treatment with TNF (10 ng/ml), SM-164 (100 nM) and z-VAD-FMK (20 μM; TSZ) in the presence or absence of RIPK1 inhibitor (RIPK1i GSK′963, 100 nM) for the indicated times. b Quantification of PI⁺ mouse dermal fibroblasts (MDF) treated as in a. c Tandem ubiquitin-binding entities (TUBE) affinity purification (AP) of ubiquitylated proteins from HT-29 cells treated with the indicated agents for the indicated timepoints. Prior to elution from the beads, samples were split in two and incubated with or without 2 μM of USP21. The presence of MLKL ubiquitylation was determined by immunoblot analysis of the eluate using α-MLKL antibody. d TUBE AP of ubiquitylated proteins from MDFs treated with the indicated agents for the indicated timepoints. * refers to non-specific bands. e Quantification of PI⁺ HT-29 cells upon treatment with TRAIL (50 ng/ml), SM-164 (100 nM) and z-VAD-FMK (20 μM; TRAIL/S/Z) for 8 h in the presence or absence of MLKL inhibitor necrosulfanamide (NSA). f TUBE AP of ubiquitylated proteins from HT-29 cells treated with TRAIL/S/Z. * refers to non-specific bands. g Quantification of PI⁺ MDF cells treated with TNF and SM-164 in presence or absence of z-VAD-FMK to induce necroptosis (TSZ) or apoptosis (TS), respectively for the indicated times. h TUBE AP of ubiquitylated proteins from MDFs treated to induce necroptosis (TSZ, 2 h) or apoptosis (TS, 3 h). * refers to non-specific bands. In a, b, e and g, n = 3 wells/group. The results are representative of those from two independent experiments with three technical replicates each. Data are presented as mean with error bars indicating standard deviation (SD). Statistical analysis shown was calculated by two-way ANOVA with Sidak’s multiple comparison test in a, b and one-way ANOVA with Sidak’s multiple comparison test in e. Source data are provided as a Source data file. c–f, h are representative of ≥1 biological replicates.