Fig. 2

TAK1 phosphorylates RIPK1 at S321. a TNFα-induced RIPK1 S321 phosphorylation is inhibited by TAK1 inhibitor. BV-2 cells were treated by TNFα (20 ng/ml) together with DMSO, Nec-1s (10 µM), TBK1 inhibitor, BX795 (1 µM) and 5Z-7 (0.5 µM). b TAK1 is required for RIPK1 S321 phosphorylation. WT and TAK1^F/F MEFs with or without Cre expression were treated with TNFα (10 ng/ml) and 5Z-7 (0.5 µM) for 15 min and examined by indicated antibodies. c Hyperphosphorylation of RIPK1 S321 in TAB2 KO MEF. TAB2 WT and KO MEFs were treated with TNFα (10 ng/ml) and the samples were collected at indicated time-points. d FLAG-RIPK1 WT and S321A were expressed and purified from 293T cells and incubated with recombinant TAK1-TAB1 or GST-IKKα or their inhibitors, 5Z-7 or PS1145, respectively. The products were analyzed by western blot using indicated antibodies. e RIPK1 S321 phosphorylation in TNF-RSC. MEF cells were treated with FLAG-TNFα (50 ng/ml) for 5 min and TNF-RSC was purified by anti-FLAG immunoprecipitation. TNF-RSC immunocomplexes were analyzed by western blot using indicated antibodies. *, nonspecific bands