Phosphomimetic S161E mutation is sufficient to confer necroptosis sensitivity in the absence of S166 phosphorylation. (A) Graphs depicting quantification of cell death in BMDMs from mice of the indicated genotypes treated with TNF or with combinations of TNF (T, 20 ng/ml), Emricasan (E, 5 µM), and the SMAC mimetic compound birinapant (S, 1 µM). Graphs show mean ± SEM of at least three independent experiments. (B) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TSE for 0, 2, and 4 h. Representative of three independent experiments. (C) Representative photographs of IKK2^E-KO (n = 7), IKK2^E-KORipk1^S166A/S166A (n = 6), and IKK2^E-KORipk1^{S161E_S166A/S161E_S166A} (n = 9) 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^S166A/S166An = 4, IKK2^E-KORipk1^{S161E_S166A/S161E_S166A}n = 4) at P8, P23, and P119. (F) Graph depicting epidermal thickness of mice with the indicated genotypes. Each dot represents one mouse. Epidermal thickness was measured at P8. 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 F5.