S161A mutation partially counteracted the inhibitory effect of S166A mutation on RIPK1 kinase activity–dependent cell death. (A) Graph depicting quantification of cell death in BMDMs from mice of the indicated genotypes treated with Emricasan (E, 5 µM), the SMAC mimetic compound birinapant (S, 1 µM), and TNF (T, 20 ng/ml). Graph shows the mean ± SEM of four independent experiments. (B) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TSE for 0, 2, and 4 h. Representative of three different experiments (C) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TSE for 0, 9, and 11 h. Representative of two independent experiments. (D) Graph depicting quantification of cell death in BMDMs from mice of the indicated genotypes treated with TAK1 inhibitor (TAK1i, 0.25 µM). Graph shows mean ± SEM of four independent experiments. (E) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TAK1i for 0, 5, and 6 h. Representative of two independent experiments. (F) Immunoblots of BMDMs from mice of the indicated genotypes stimulated with TAK1i for 0, 10, and 12 h. Representative of two independent experiments. Source data are available for this figure: SourceData F4.