Fig. 2: RIPK1 kinase acted as the upstream effector driving Ripoptosome assembly.

a Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in WT, RIPK1 KO, or ZBP1 KO cells infected with RS218 (MOI = 1). b Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in hBMECs expressing RIPK1 WT or D138N under RS218 infection (MOI = 1). c Immunoblot of RIPK1 total protein and phosphorylation levels at S161 and S166 in WT, TNFR1 KO, TLR4 KO, or double KO cells post RS218 infection (MOI = 1). Blots for GAPDH served as loading controls. d Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in hBMECs expressing RIPK1 WT, S161N, or S166N cells (MOI = 1). e, f In WT, TNFR1 KO, TLR4 KO, or double KO cells, the cytotoxicity was assessed by LDH release (e, n = 3 biologically independent samples), and cell death kinetics were measured by PI uptake (f, n = 3 biologically independent samples). g Co-IP analysis of RIPK1 with TNFR1 and TLR4 interactions in WT and ZBP1 KO cells. h Co-IP of pro-caspase-8 with RIPK1, RIPK3, ZBP1, and ASC in WT, TNFR1 KO, TLR4 KO or double KO cells (MOI = 1). i Immunoblot of RIPK1 total protein and phosphorylation levels at S161 in WT or ZBP1 KO cells. Blots for GAPDH served as loading controls. All experiments were representative of at least three independent experiments with similar results. Bars indicated the mean plus SD. Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test, with P-values denoted as follows: **** P < 0.0001, *** P < 0.0005, ** P < 0.005, * P < 0.05, n.s., no significant difference. Exact P-values were provided in the source data. Source data are provided as a Source Data file.