Figure 5

CRBN inhibits ubiquitination of TAB2. (a) HEK293T cells were transfected with mock, Myc-CRBN, Flag-TAB2, or HA-Ub, as indicated. At 38 h after transfection, transfected cells were extracted, immunoprecipitated with anti-Flag antibody, and then immunoblotting was performed with anti-Flag, anti-Myc, or anti-HA antibodies. (b) HEK293T cells were transfected with mock, HA-Ub, and Flag-TAB2 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. (c) CRBN^+/+ or CRBN^−/− MEF cells were stimulated without or with LPS for different periods of time, as indicated, then subjected to extraction, and western blotting was performed with the antibodies indicated on the left. The band intensity of p-IKKα/β was analyzed with Image J (bottom). All error bars represent S.D. of the mean from three independent experiments *P<0.05 versus without LPS. (d) TRAF6^KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6 wt, Flag-TAB2, 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. (e) TRAF6^KD and Ctrl THP-1 cells were transfected with mock, Flag-TRAF6 wt, Flag-TAB2, or HA-CRBN vectors, as indicted, and treated with or without LPS (200 ng/ml) for 9 h, and 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