Fig. 6

S321A mutation promotes RIPK1 activation. a RIPK1 S166 phosphorylation in S321A(A/A) mutant. WT and S321A(A/A) MEFs were treated with TNFα (50 ng/ml), CHX (1 µg/ml), zVAD (20 µM) and Nec-1s (10 µM) as indicated for 2 h. b S321A mutation augments RIPK1 K63 ubiquitination in response to TNFα/CHX treatment. MEFs were treated with TNFα (50 ng/ml) and CHX (1 µg/ml). Lysates under denaturing condition were collected at indicated time-points, immunoprecipitated with K63 antibody and detected with RIPK1 antibody. c Enhanced MLKL phosphorylation in S321A(A/A) cells in response to TNFα/CHX/zVAD. MEFs were treated with TNFα (10 ng/ml), CHX (1 µg/ml), zVAD (20 µM) for 2 and 4 h. Arrow, phosphorylated MLKL. d Earlier induction of RIPK1-RIPK3 interaction in S321A(A/A) MEFs induced by TNFα/CHX/zVAD. WT and RIPK1 S321A(A/A) MEFs were treated as a and co-immunoprecipitation was performed with RIPK3 antibody. e Stronger interaction between FADD and RIPK1 in RIPK1 S321A(A/A) MEFs induced by TNFα/CHX was suppressed by Nec-1s. The cells were treated with TNFα (50 ng/ml) and CHX (1 µg/ml) with or without Nec-1s (20 µM) for 4 h. Co-immunoprecipitation was performed with FADD antibody. *, nonspecific band