USP25 has a dose-dependent deubiquitinating activity <i>in</i><i>vivo</i>.

<p>(A) HEK-293T cells grown in 60-mm dishes were transfected with Flag-tagged Ub expression plasmids (1 μg), along with increasing quantities (0.25, 0.5, 1, or 2 μg) of plasmid encoding USP25 using Lipofectamine 2000. Cell lysates were prepared at 30 h post-transfection and analyzed for Ub-conjugated proteins by Western blotting with an anti-Flag antibody. Western blotting with anti-HA antibodies shows expression of USP25, and Western blotting for beta-actin served as a protein loading control. (B, C) USP25 effectively cleaved both K48 and K63 Ub linkages <i>in </i><i>vivo</i>. The experiment was performed similarly to that described for panel A except that HA-K48-Ub or HA-K63-Ub was used in lieu of Flag-Ub and different expression plasmids of USP25 were used. (D) Mapping of the putative sites are associated with the DUB activity of USP25. Black boxes indicate conserved residues tested in this study. The sequences were derived from GenBank entries with the following accession numbers: USP2, NM_004205; USP4, NM_003363; USP11, NM_004651; USP17, NM_201402 and XM_352721; USP20, NM_001110303; USP21, NM_001014443; and USP25, NM_013396. (E) Cysteine 178 (C178) and histidine 607 (H607) are deubiquitination active sites of USP25. Expression vectors encoding pcDNA3.1-Flag-Ub and control vectors or expression vectors encoding HA-USP25-WT, -C178A and -H607A were co-transfected into HEK-293T cells. USP25-WT and deletion mutant proteins in cell lysates were immunoprecipitated with anti-HA antibodies under denaturing conditions and immunoblotted with anti-Flag antibodies to detect the presence of Ub-conjugated proteins. Western blotting for beta-actin served as a protein loading control.