S161 and S166 cooperate to regulate RIPK1 kinase activation. (A) Schematic depicting the possible combinations of RIPK1 dimers in Ripk1^{S161N_S166A/D138N} and in Ripk1^S166A/D138N mice. Residue 166 on RIPK1 is labeled. (B) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TNF (T, 20 ng/ml), the SMAC mimetic compound birinapant (S, 1 µM), and Emricasan (E, 5 µM) (TSE) for 0, 2, 4, and 6 h. Representative of three independent experiments. (C) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TSE for 0, 7, or 9 h. Representative of two independent experiments. (D) Graph depicting quantification of cell death in BMDMs from mice of the indicated genotypes treated with TSE. Graph shows the mean ± SEM of seven independent experiments. (E) Immunoblots of BMDMs from mice of the indicated genotypes treated with TAK1 inhibitor (TAK1i, 0.25 µM) for 0, 13, and 15 h. Representative of two independent experiments. (F) Graph depicting quantification of cell death in BMDMs from mice of the indicated genotypes treated with TAK1i (0.25 µM). Graph shows the mean ± SEM of four independent experiments. Source data are available for this figure: SourceData F7.