Autophosphorylation at S161 drives RIPK1 kinase activity–dependent necroptosis. (A) Graphs depicting quantification of cell death in BMDMs from mice of the indicated genotypes treated with combinations of Emricasan (E, 5 µM), the SMAC mimetic compound birinapant (S, 1 µM), TNF (T, 20 ng/ml), and LPS (L, 100 ng/ml). Graphs show mean ± SEM from at least four independent experiments. (B) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TSE for 0, 2, or 4 h. Representative of three independent experiments. (C) Representative photographs of control (n = 8), IKK2^E-KO (n = 7), IKK2^E-KORipk1^S161A/S161A (n = 5), and IKK2^E-KORipk1^S161N/S161N (n = 5) mice. (D) Kaplan–Meier survival curve of mice with the indicated genotypes. (E) Representative images of sections from back skin of mice with the indicated genotypes stained with H&E or immunostained for K6 (scale bars = 100 µm; control n = 7, IKK2^E-KOn = 7, IKK2^E-KORipk1^S161A/S161An = 4, and IKK2^E-KORipk1^S161N/S161Nn = 4). (F) Graph depicting epidermal thickness of mice with the indicated genotypes. Each dot represents one mouse. Mean ± SEM are shown. Statistical significance was determined using Kruskal–Wallis test. (G) Graphs depicting relative mRNA expression of the indicated cytokines in RNA from whole-skin tissue of mice of the indicated genotypes measured by qRT-PCR. Each dot represents one mouse. Mean ± SEM are shown. Statistical significance was determined using Kruskal–Wallis test. Control mice include Ikk2^FL/FLK14Cre^WT/WT or Ikk2^FL/WTK14Cre^Tg/WT littermates with WT or mutant Ripk1 alleles. Source data are available for this figure: SourceData F1.