Mimicking S161 phosphorylation can compensate for the loss of RIPK1 kinase activity in initiating TNF-induced mouse death. (A–C) Survival curves and body temperature of 8- to 12-wk-old male littermate mice after TNF injection (400 μg/kg i.v. for Ripk1^+/+ and Ripk1^K45A/K45A mice; 50 μg/kg i.v. for Ripk1^S161E/S161E and Ripk1^{K45A+S161E/K45A+S161E} mice). Before TNF injection in C, Ripk1^S161E/S161E mice were intragastrically administered with Necrostatin 1S (Nec-1s) or sodium carboxymethyl cellulose (CMC-Na). Mouse survival is presented as a Kaplan–Meier plot, and the log-rank test is performed. ns, P ≥ 0.05; ***P < 0.001. Data of body temperature are presented as mean ± SD. (D) Primary BMDMs from Ripk1^+/+, Ripk1^K45A/K45A, Ripk1^S161E/S161E, and Ripk1^{K45A+S161E/K45A+S161E} mice were treated with TNF (10 ng/ml) + zVAD (20 μM) for the indicated time points. Survival of cells from three mice of each genotype was measured. All mice were 8- to 12-wk-old male littermates. Data are presented as mean ± SD. (E) Cell lysates of primary BMDMs of indicated genotypes treated with or without TNF (10 ng/ml) + zVAD (20 μM) for 6 h were analyzed by immunoblotting to detect proteins as indicated. The above experiments were independently performed twice. Source data are available for this figure: SourceData F4.