Fig. 1. S166 phosphorylation drives RIPK1-dependent cell death.

a BMDMs from mice of the indicated genotypes were treated with FLAG-hTNF for 0 or 5 min and FLAG-immunoprecipitates were analyzed with the indicated antibodies. Representative data of two independent experiments is shown. b BMDMs from mice of the indicated genotypes were treated with a combination of TNF (T; 10 ng ml⁻¹), Z-VAD-FMK (Z) and SMAC mimetic (S (Birinapant); 1 µM) in the presence or absence of Nec-1s. c Primary dermal fibroblasts from mice of the indicated genotypes were treated with combinations of TNF (T; 20 ng ml⁻¹), Z-VAD-FMK (Z) and SMAC mimetic (S (Birinapant); 1.5 µM) in the presence or absence of Nec1s. d Primary lung fibroblasts from mice of the indicated genotypes were treated with combinations of TNF (T; 20 ng ml⁻¹), Z-VAD-FMK (Z) and SMAC mimetic (S (Birinapant); 1 µM) in the presence or absence of Nec-1s. e Primary dermal fibroblasts from mice of the indicated genotypes were treated with combinations of TNF (T; 20 ng ml⁻¹) and SMAC mimetic (S (Birinapant); 1.5 µM) in the presence or absence of Nec-1s. f Primary lung fibroblasts from mice of the indicated genotypes were treated with combinations of TNF (T; 20 ng ml⁻¹) and SMAC mimetic (S (Birinapant); 1 µM) in the presence or absence of Nec-1s. g Primary lung fibroblasts from mice of the indicated genotypes were treated with TNF (20 ng ml⁻¹) in the presence or absence of cycloheximide (CHX, 1 μg ml⁻¹) and Nec1-s. h BMDMs were treated with LPS (L; 100 ng ml⁻¹) and Z-VAD-FMK (Z) in the presence or absence of Nec-1s. i BMDMs were treated with Poly(I:C) (P; 0.5 μg ml⁻¹) and Z-VAD-FMK (Z) in the presence or absence of Nec-1s. b–i Cell death was measured using an IncuCyte as described. Graphs represent four independent experiments in b, d, f and h; three independent experiments in c, e and i and two independent experiments in g. Source data for b–i are provided as a source data file.