Fig. 7

Hyperactivation of RIPK1 mediated by TAK1 sensitizes cells to necroptosis. a S321E mutation suppresses RIPK1 cleavage and RIPK1-FADD interaction induced by TNFα/CHX. The cells were treated with TNFα (50 ng/ml) and CHX (1 µg/ml) for 2 or 4 h. Co-immunoprecipitation was performed with FADD antibody. b TNFα induces RIPK1 S332/334 phosphorylation. RGC-5 cells were treated TNFα (10 ng/ml) with or without 5Z-7 (0.5 µM). RIPK1 was isolated by immunoprecipitation and detected by p-RIPK1 S332/334 and RIPK1 total antibodies. c RIPK1 S321/332/334/336A (AAAA) mutation blocks TNFα-induced S332/334 phosphorylation. FLAG-RIPK1 WT, AAAA mutant or empty vector was transiently expressed in RGC-5 cells and treated with TNFα (10 ng/ml) and 5Z-7 (0.5 µM) for 15 min. FLAG-RIPK1 was purified by anti-FLAG immunoprecipitation and detected by p-RIPK1 S332/334 and RIPK1 total antibodies. d RIPK1 kinase activity and its interaction with RIPK3 were induced by RIPK1 S321/332/334/336E (EEEE) mutant overexpression. RGC-5 RIPK1 KO cells were transfected with RIPK1 WT and mutants. 30 h after transfection, the cells were lysed and immunoprecipitated with RIPK1 p-S166 or RIPK3 antibodies and blotted with RIPK1 total antibody. e Transient expression of RIPK1 EEEE mutant promotes MLKL phosphorylation in RGC-5 cells. RGC-5 cells were transfected with FLAG-RIPK1 variants and samples were collected 24 h after transfection. f Spontaneous necroptosis in response to the expression of RIPK1 EEEE mutant. RGC-5 RIPK1 KO cells with or without RIPK3 knockdown were transfected with RIPK1 WT or mutants. GSK843 (10 µM)/zVAD (20 µM) was added as indicated. Cell death was measured by ToxiLight assay 24 h after transfection. *, nonspecific bands. Error bar, s.e.m. ***, t-test P < 0.001; n.s., not significant. Three independent repeats were included in each data point