Figure 4: TNF promotes binding of Pellino3 to RIP1 in a FHA domain-dependent manner but not as part of complex I.

(a,b) Hela cells were treated in the absence or presence of TNF (50 ng ml⁻¹) and cycloheximide (CHX) (10 μg ml⁻¹) for the indicated times. Cells were also treated with the pan-caspase inhibitor ZVAD (20 μM) to prevent full activation of caspase 8 and cell death. Cell lysates were immunoprecipitated with anti-RIP1 (a) or anti-caspase 8 (b) antibodies. Mouse IgG and goat IgG were used for respective control immunoprecipitations. The immunoprecipitates were subsequently analysed for co-precipitated Pellino3, RIP1, caspase 8 and FADD by western blotting. The expression levels of Pellino3, RIP1, caspase 8 and FADD proteins were measured in whole-cell lysates (input) by western blotting. The precipitated IgG heavy chain (IgGH) is also indicated by an arrow. (c) Hela cells were treated in the absence or presence of TNF (50 ng ml⁻¹) for indicated times. Cell lysates were immunoprecipitated with anti-TNFR1 antibody. The immunoprecipitates were subsequently analysed for co-precipitated Pellino3, RIP1, TRAF2 and TNFR1 by western blotting. The expression levels of Pellino3, RIP1, TRAF2 and TNFR1 proteins were measured in whole-cell lysates (input) by western blotting. (d–f) HEK293 cells were transfected with an empty vector (EV) or expression constructs encoding (d) myc-taggged Pellino3s or Pellino3l, (e) FHA domain mutant forms of Pellino3s(R131A/S161A) (Pellino3sFHAm) or Pellino3l(R155A/S185A) (Pellino3lFHAm) or (f) RING-like domain mutant forms of Pellino3s (C360A/C363A) (Pellino3sE3m) and Pellino3l (C384A/C387A) (Pellino3lE3m). Cells were then treated in the absence or presence of TNF (50 ng ml⁻¹) and cycloheximide (CHX) (10 μg ml⁻¹) for indicated times. Cells were also treated with the pan-caspase inhibitor ZVAD (20 μM) to prevent full activation of caspase 8 and cell death. Cell lysates were immunoprecipitated with an anti-myc antibody and immunoprecipitates were subsequently analysed for co-precipitated RIP1, caspase 8, FADD and myc-tagged Pellino proteins by western blotting. The precipitated IgG light chain (IgGL) is indicated by an arrow. The expression levels of RIP1, caspase 8, FADD and myc-tagged Pellino proteins were measured in whole-cell lysates (input) by western blotting. Full-length images of immunoblots are shown in Supplementary Fig. S11.