FIGURE 5.

Iron induces Lys⁶³-polyUb of TRAF6. A, cell lysates collected from iron-treated hepatic macrophages at different times were immunoprecipitated (IP) with anti-Lys⁶³-Ub and immunoblotted (WB) with anti-TRAF6. An arrow indicates polyubiquitinated TRAF6 (Ub-TRAF6) which increases after iron treatment. B, reciprocally, the cell lysates were immunoprecipitated with anti-TRAF6 and immunoblotted with anti-Lys⁶³-Ub, demonstrating similar results. C, expression of the dominant negative mutant of Ub (Ub-K63R) but not wild-type Ub (Ub-Wt) eliminates the formation of Ub-TRAF6 in response to iron. D, the Ub-K63R also blocks iron-induced activation of TAK1 as assessed by immunoblotting of p-TAK1 (Thr^184/187). E, SOD attenuates iron-induced Lys⁶³-Ub of TRAF6 and pERK1/2.