Fig. 1

TNFα induces RIPK1 phosphorylation at S321. a Alignment of amino acid sequences in the relevant part of RIPK1 intermediate domain from indicated mammalian species. S321, S332, S334 and S336 as marked by arrowheads are highly evolutionarily conserved. b S321 of RIPK1 is transiently phosphorylated after TNFα treatment. MEF cells were treated with TNFα (10 ng/ml) and the samples were collected at indicted time-points. The cell lysates were analyzed by western blot. c Anti-p-S321-RIPK1 antibody specifically recognizes RIPK1 S321 phosphorylation. L929 cells were treated with TNFα (20 ng/ml) for 5 min. The cell lysates were subjected to immunoprecipitation by either RIPK1 antibody or IgG. Both lysates and IP samples were examined by western blot using anti-p-S321-RIPK1 and RIPK1 total antibodies. d, e TNFα induces RIPK1 S321 phosphorylation in murine primary BMDM and microglia cells. BMDM d and primary microglia e were pre-incubated with DMSO or TAK1 inhibitor, 5Z-7 (0.5 µM) for 30 min and treated with TNFα (10 ng/ml). Samples were collected at indicated time-points. *, nonspecific bands