Triad1 inhibits Gfi1 ubiquitination. (A) Gfi1-GFP and Flag-Ub were cotransfected with or without Triad1. Gfi1 was immunoprecipitated with α-GFP. Ubiquitinated Gfi1 or ubiquitinated proteins bound to Gfi1 were detected by α-Flag staining. The high molecular smear suggests that ubiquitinated proteins are bound to Gfi1, or that Gfi1 itself is ubiquitinated (→ marks molecular weight of unmodified Gfi1-GFP). The coexpression of Triad1 severely reduces the amount of ubiquitinated Gfi1 or ubiquitinated proteins bound to Gfi1. α-GFP staining of whole-cell extract (wce) was used to check for equal expression. (B) COS cells were transfected as indicated. Ubiquitinated proteins were isolated with His-select beads under denaturing conditions (6 M guanidium chloride). The high molecular smear represents polyubiquitinated forms of Gfi1. Coexpression of Triad1 or the H158A point mutant severely inhibited the ubiquitination of Gfi1. Incubation of cells with MG132 resulted in an accumulation of ubiquitinated forms of Gfi1, indicating that polyubiquitination of Gfi1 results in proteasomal degradation. Triad1 also inhibited the ubiquitination of Gfi1 in the presence of the proteasome inhibitor MG132. *Nonspecific interaction of Gfi1 with the His-select beads. (C) In vivo ubiquitination assay of endogenous proteins with His-Ub. Selection of ubiquitinated proteins (His-select beads) followed by an α-His staining showed that overall ubiquitination is not inhibited by Triad1. The α-ubiquitin staining of whole-cell extract (wce) in the bottom panel showed equal expression of His-tagged ubiquitin. The lower band is endogenous ubiquitin; the slower migrating band is His-Ub.