Figure 5: Pellino3 inhibits formation of Complex II in a FHA-dependent but RING-like domain-independent manner.
From: Pellino3 targets RIP1 and regulates the pro-apoptotic effects of TNF-α
HEK293 cells were transfected with pcDNA3.1 empty vector (EV) or pcDNA3.1 encoding myc-tagged Pellino3s (P3s), Pellino3s(R131A/S161A)-myc (P3sFHAm), Pellino3s(C360A/C363A)-myc (P3sE3m) and flag-Pellino3sΔC(1–313aa) (P3sΔcm). Transfected cells were treated with human TNF (40 ng ml−1), cycloheximide (CHX) (10 μg ml−1) and ZVAD(20 μM) for 4 h. Cell lysates were immunoprecipitated with an anti-RIP1 antibody and immunoprecipitates were subsequently analysed for co-precipitated RIP1, FADD, Myc, Flag and caspase 8 by western blotting. The expression levels of RIP1, FADD, caspase 8 and Pellino3 were measured in whole-cell lysates (input) by western blotting. Full-length images of immunoblots are shown in Supplementary Fig. S12.
