FIG. 8.

TRAF6 regulates TRAF2-dependent CD40-mediated signaling. (A) TRAF2 interacts with exogenously expressed TRAF6 in the presence of CD40mT6. HEK 293 cells were transiently transfected with FLAG-tagged TRAF6 in the presence or absence of CD40mT6, and 24 h later cells were lysed. Lysates (50 μg) were immunoblotted with anti-Flag or anti-CD40 to confirm expression of transfected cDNAs (i). Lysates (750 μg) were also immunoprecipitated with anti-TRAF2 MAb and immunoblotted with either anti-FLAG or anti-TRAF2 polyclonal antibody, in parallel with 50 μg of whole-cell lysate (ii), or immunoprecipitated with anti-FLAG MAb and immunoblotted with either anti-TRAF2 or anti-TRAF6 polyclonal antibody, in parallel with 50 μg of whole-cell lysate (iii). (B) TRAF2 interacts with exogenously expressed TRAF6 in the presence of CD40Δ(216-239). HEK 293 cells were transiently transfected with FLAG-tagged TRAF6 in the presence or absence of CD40Δ(216-239), and 24 h later cells were lysed. Lysates (750 μg) were immunoprecipitated with anti-TRAF2 MAb and immunoblotted with either anti-FLAG or anti-TRAF2 polyclonal antibody, in parallel with 50 μg of whole-cell lysate. (C) TRAF6 and TRAF2 coimmunoprecipitate in CD40L-stimulated carcinoma cells carrying a mutated CD40 that does not directly bind TRAF6. HeLa-CD40mT6 cells were treated with 0.1 μg/ml CD40L for various time intervals or left untreated, as indicated. Lysates were then immunoprecipitated with an anti-TRAF2 MAb and immunoblotted with either anti-TRAF6 or anti-TRAF2 polyclonal antibody. Similar results were obtained in two additional independent experiments. Ig, immunoglobulin heavy chain. (D) TRAF6 and TRAF2 do not coimmunoprecipitate in CD40L-stimulated carcinoma cells carrying a mutated CD40 that does not directly bind TRAF2/TRAF3. HeLa-CD40mT2/T3 (lanes 3 to 5) or, as a control, HeLa-CD40mT6 (lanes 1 to 2) cells were treated with 0.1 μg/ml CD40L for various time intervals or left untreated, as indicated. Lysates were then immunoprecipitated with an anti-TRAF2 MAb and immunoblotted with either anti-TRAF6 or anti-TRAF2 polyclonal antibody. (E) HeLa-CD40mT6 cells were transfected with siRNA targeting TRAF6 or the unrelated luciferase gene product. Lysates were analyzed for TRAF6, TRAF2, or β-actin levels by immunoblotting. (F) RNAi-mediated knock-down of TRAF6 expression results in diminished CD40-induced NF-κB, JNK, and p38 activation in HeLa-CD40mT6 epithelial cells. Following transfection with TRAF6 or luciferase siRNA, HeLa-CD40mT6 cells were stimulated with 0.1 μg/ml CD40L or left untreated, as indicated. Lysates were isolated and analyzed for the expression of IκBα and of the phosphorylation and activation of p38 and JNK, respectively. Data are representative of three independent experiments. IP, immunoprecipitate; IB, immunoblot; WCL, whole-cell lysate.