Phosphomimetic S161E mutation does not sensitize cells to RIPK1 kinase activity–dependent apoptosis in the absence of S166 phosphorylation. (A) Graphs depicting quantification of cell death in BMDMs from mice of the indicated genotypes treated with TAK1 inhibitor (TAK1i, 0.25 µM) alone and with a combination of TAK1i and Emricasan (5 µM). Graphs show mean ± SEM of three independent experiments. (B) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TAK1i with or without Emricasan (E, 5 µM). Representatives of two independent experiments. (C) Kaplan–Meier survival curve of mice with the indicated genotypes. (D) Representative images of sections from back skin of mice with the indicated genotypes stained with H&E or immunostained for CC3 (scale bars = 100 µm; control n = 5, Sharpin^cpdm/cpdmn = 10, Sharpin^cpdm/cpdmRipk1^S166A/S166An = 4, Sharpin^cpdm/cpdmRipk1^{S161E_S166A/S161E_S166A}n = 4). (E) Graph depicting measurement of epidermal thickness of mice with the indicated genotypes. Each dot represents one mouse. (F) Graph showing the average amount of CC3⁺ cells per optical field in mice with the indicated genotypes. Mean ± SEM are shown. Each dot represents one mouse. 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. Means ± SEM are shown. Statistical significance was determined using Kruskal–Wallis test. Control mice include Sharpin^cpdm/WT or Sharpin^WT/WT littermates with WT or mutant Ripk1 alleles. Source data are available for this figure: SourceData F6.