Figure 7. TRIF and IAPs regulate LPS-induced ubiquitylation of RIPK3 and MLKL.

(a) WT, Myd88^−/− and Trif^−/− BMDM were cultured with neutralizing antibodies to TNF (anti-TNF [XT-22] 20 μg ml⁻¹), isotype control antibodies (IC [GL113] 20 μg ml⁻¹) or Nec-1 (50 μM), and primed for 2 h with LPS, as indicated. Cells were then cultured with Cp.A (500 nM), and IL-1β levels were assayed in supernatants by ELISA at 6 h. n=3 mice per group, mean+s.e.m., representative of one of three experiments. (b) WT and Tnf^−/− BMDM were primed for 3 h with LPS, and treated with Nec-1 (50 μM) as indicated. Cells were then cultured with Cp.A (500 nM), and after 6 h IL-1β secretion was assayed by ELISA. n=3 mice per genotype, mean+s.e.m., one of three experiments. (c,d) WT, Myd88^−/− and Trif^−/− BMDM were cultured with neutralizing antibodies to TNF (anti-TNF [XT-22] 20 μg ml⁻¹), isotype control antibodies (IC [GL113] 20 μg ml⁻¹) or Nec-1 (50 μM), and primed for 2 h with LPS, as indicated. Cells were then cultured with Cp.A (500 nM) for 24 h, and (c) IL-1β levels assayed in supernatants by ELISA and (d) cell death (% Dead cells) assessed by PI and FACS. n=3 mice per group, mean+s.e.m., representative of one of three experiments. (e) WT BMDM were treated with 50 ng ml⁻¹ LPS (30 and 60 min) or 100ng ml⁻¹ TNF (20 min) and ubiquitylated proteins were isolated by TUBE and analyzed by immunoblot. One of two experiments. (f) WT, Myd88^−/−, and Trif^−/− BMDM were treated with LPS (100 ng ml⁻¹) for 60 min and endogenous ubiquitylated proteins isolated by TUBE and analyzed by immunoblot. One of three experiments. (g) WT and Trif^−/− BMDM were pre-incubated with Q-VD-OPh (20 μM) for 40 min, cultured with Cp.A 500 nM and subsequently treated with LPS (50 ng ml⁻¹) for 60 or 190 min as indicated. Endogenous ubiquitylated proteins were isolated by TUBE and analyzed by immunoblot. One of three experiments.