Fig. 3.

TNF-α induces Miz1 K48-linked polyubiquitination of TRAF2-associated Miz1. (A) TRAF2 was immunoprecipitated from WT fibroblasts that have been pretreated without or with MG-132 (50 μM, 2 h) and then treated with TNF-α (5 ng/mL) for various periods of time, as indicated. TRAF2-coimmunoprecipitated Miz1 was detected by immunoblotting using anti-Miz1 antibody. Miz1*, upshifted Miz1. (B and C) TNF-α–induced degradation of Miz1 was dependent on TRAF2. (D and F) TNF-α–induced polyubiquitination of Xpress-Miz1 in the presence of cotransfected HA-Ub (D), or HA-Ub, HA-K63O-Ub, or HA-K48O-Ub (F) (5 μg each) was analyzed. (E) TNF-α–induced ubiquitination of Miz1 in the presence of MG-132 (see Methods for details).