Fig. 7. S166 phosphorylation enhances RIPK1 kinase activity.

a Scheme depicting the possible combinations of RIPK1 dimers in Ripk1^D138N/S166A mice. Residue 166 on RIPK1 is indicated. b, BMDMs from mice of the indicated genotypes were treated with a combination of TNF (T; 10 ng ml⁻¹), SMAC mimetic (S (Birinapant), 1 μM) and Z-VAD-FMK (Z) for 0, 2 or 4 h and lysates were analyzed by immunoblot with the indicated antibodies. Blots shown are representative of three independent experiments. *Non-specific band. c Primary lung fibroblasts from mice of the indicated genotypes were treated with a combination of TNF (T; 20 ng ml⁻¹), SMAC mimetic (S (Birinapant), 1 μM) and Z-VAD-FMK (Z) for 0, 3 or 5 h and lysates were analyzed by immunoblot with the indicated antibodies. Blots shown are representative of three independent experiments. d BMDMs from mice of the indicated genotypes were treated with combinations of TNF (T; 10 ng ml⁻¹), SMAC mimetic (S (Briniapant), 1 μM) and Z-VAD-FMK (Z) in the presence or absence of Nec-1s. Cell death was measured using IncuCyte. Graph shows mean of technical triplicates. Data are representative of three independent experments. For purpose of comparability the data of the genotypes WT, Ripk1^S166A/S166A and Ripk1^D138N/D138N shown here are identical to those in Fig. 1b. e BMDMs were treated with a combination of TNF (T; 10 ng ml⁻¹), SMAC mimetic (S (Birinapant), 1 μM) and Z-VAD-FMK (Z) for 0, 2 or 4 h. FADD immunoprecipitates and total cell lysates (input) were analyzed by immunoblot using the indicated antibodies. f BMDMs were treated with a combination of TNF (T; 10 ng ml⁻¹), SMAC mimetic (S (Birinapant), 1 μM) and Z-VAD-FMK (Z, 50 μM) for 0, 2 or 4 h. Caspase-8 immunoprecipitates and input were analyzed by immunoblot using the indicated antibodies. Source data for d are provided as a source data file.