Figure 4.

CRBN interacts with TRAF6 and inhibits the ubiquitination of TRAF6. (a) HEK293T cells were transfected with mock, HA-CRBN, or Flag-TRAF6, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then immunoblotting was performed with anti-Flag or anti-HA antibodies. (b) A schematic presentation of TRAF6 wt and its truncated mutants used in this study: TRAF6 110-522, TRAF6 260-522, and TRAF6 349-522. (c) HEK293T cells were transfected with HA-CRBN, Flag-TRAF6 wt, Flag-TRAF6 110-522, Flag-TRAF6 260-522, or Flag-TRAF6 349-522, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag or anti-HA antibodies. (d) HEK293T cells were transfected with mock, Myc-CRBN, Flag-TRAF6, or HA-Ub, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. (e) HEK293T cells were transfected with mock, HA-Ub, and Flag-TRAF6 in the absence or presence of different concentrations of Myc-CRBN. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with an anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. (f) THP-1 cells were infected with a lentivirus containing shRNA targeted to human TRAF6 or control shRNA sequences. Two weeks post-infection, endogenous expression of TRAF6 protein was analyzed in TRAF6^KD and control (Ctrl) THP-1 cells. (g) TRAF6^KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6, or HA-CRBN vectors, as indicted, together with pBIIx-luc and Renilla luciferase. Twenty-four hours after transfection, cells were untreated or treated with LPS (200 ng/ml) for 6 h and then analyzed for luciferase activity. Results are expressed as the fold induction in luciferase activity relative to that in untreated cells. All error bars represent S.D. of the mean from triplicate samples. *P<0.05. (h) TRAF6^KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6, or HA-CRBN vectors, as indicated, and treated with or without LPS (200 ng/ml) for 9 h, then, production of IL-6 was analyzed by ELISA. All error bars represent S.D. of the mean from triplicate samples. *P<0.05 and **P<0.01