(A) The TRAF domain is required for TRAF4 interacting with TRAF2. Wild type, ΔR and ΔT mutants were transfected into MCF7 cells. The cell lysates were then incubated with TRAF2 antibody and protein A/G agarose beads. Western blot was performed to analysis the mutants that co-immunoprecipitation by TRAF2 antibody. (B) The Zinc fingers domain is indispensable for TRAF4 binding to TRAF2 in MCF7. Flag tagged ΔZn mutant was transfected into MCF7 cells, co-immunoprecipitation using TRAF2 or Flag antibody and Western blot were performed as before. Both TRAF2 and Flag could be dragged down by each other. (C) TNFa induces phosphorylation and activation of p70s6k. MCF7 cells were treated with TNFa (20ng/ml) for different time. Western blot was to detect p-p70s6k protein level. The phosphorylation of p70s6k peaked at 15minutes and maintained until 30 minutes. (D, E) Both TRAF4 and TRAF2 play critical role in the activation of p70s6k/S6 pathway induced by TNFa. Koncking down TRAF2 or TRAF4 significantly reduced the activation of p70s6k/S6 pathway induced by TNFa. (F) TRAF2 lies upstream of TRAF4 in TNFa-induced p70s6k pathway activation. The distribution of TRAF2 and TRAF4 after TNFa treatment were detected by immunofluorrescence staining. TRAF2 had obviously distribution of cytosolic side near the cell membrane, and co-localization of TRAF4 and TRAF2 was obviously found in this section after TNFa stimulation. TRAF2 (red), TRAF4 (green), bar, 10 μm. (G) The interactions between mTOR and p70s6k, p70s6k and TRAF2, TRAF2 and TRAF4 were enhanced after TNFa treatment. TRAF4 expression vector was employed into MCF7 together with or without TNFa. The interaction between them were detected by co-immunoprecipitation and Western blotting. After TNFa stimulation, more p70s6k interacted with mTOR, more TRAF4 interacted with p70s6k, more TRAF4 interacted with TRAF2. The relative expression levels of TRAF4 binding to p70s6k, p70s6k binding to mTOR, TRAF4 binding to TRAF2 were calculated as before. Data was presented as mean ± SD from three independent experiments. (*p < 0.05).