Figure 10: Pellino3-deficient MEFs show enhanced levels of complex II but no effect on complex I formation in response to TNF.

(a) MEFs were treated with TNF (40 ng ml⁻¹) for the indicated times. Cell lysates were immunoprecipitated with an anti-TNFR1 antibody. Immunoprecipitates and cell lysates (‘input’) were probed for levels of RIP1, TRAF2, TAK1, NEMO and TNFR1 by immunoblotting. (b) MEFs were treated with TNF (40 ng ml⁻¹) for the indicated times and followed by immunoprecipitation of endogenous RIP1. Immunoprecipitates and cell lysates (‘input’) were probed using anti-ubiquitin and -RIP1 antibodies. (c) MEFs were isolated from Pellino3^+/+ and Pellino3^−/− embryos and treated with TNF (1.5 μg ml⁻¹) for the indicated times. Complex II was immunoprecipitated using an anti-FADD and analysed for co-precipitated RIP1 and caspase 8. The whole-cell lysates (input) were immunoblotted using RIP1, caspase 8, FADD, cleaved PARP, cleaved caspase 3 and β-actin antibodies. Cleaved forms of caspase 8 are indicated by arrows (d) Immortalized BMDMs from Pellino3^+/+ and Pellino3^−/− mice were treated in the absence (control) or presence of murine TNF (40 ng ml⁻¹) and Smac mimetic (10 μM) for 16 h. Cells were also treated with ZVAD to prevent activation of caspase 8 and cell death. Cell lysates were immunoprecipitated using an anti-FADD antibody and analysed for co-precipitated RIP1 and caspase 8. The whole-cell lysates (input) were immunoblotted using RIP1, caspase 8, pan-cIAP and FADD antibodies. Full-length images of immunoblots are shown in Supplementary Fig. S17.