Fig. 3. USP25 interacted with and deubiquitinated HBO1.

(A) THP-1 cell lysates were co-immunoprecipitated with HBO1 antibody. The immunoprecipitates were analyzed with DUBs immunoblotting (see table 1). (B) THP-1 cells were transfected with USP25-DsiRNA sets and scrambled-DsiRNA for 72 h. The cell lysates were analyzed with USP25, HBO1 and β-actin immunoblotting. The densitometry results were plotted in lower panel. (C) THP-1 cells were transfected with scrambled-DsiRNA, USP25-DsiRNA or USP25-DsiRNA plus pCMV6-XL5/USP25 plasmid for 72 h. Cell lysates were subjected to HBO1, USP25, or β-actin immunoblotting. The densitometry results were plotted in lower panel. (D) Human primary macrophages were harvested, cell lysates (1mg of total protein) were subjected to HBO1 immunoprecipitation followed by USP25 and HBO1 immunoblotting as indicated. (E–F) THP-1 cells were transfected with USP25-DsiRNA-3 or scrambled-DsiRNA for 72 h. Equal amount of cell lysates (1mg of total protein) were subjected to HBO1 immunoprecipitation followed by immunoblotting as indicated. The efficacy of USP25 knockdown in each group was determined by immunoblotting as indicated. The densitometry results of E were plotted in F. (G, H) THP-1 were treated with MG132 for 30 min, cell lysates were immunoprecipitated with a HBO1 antibody and incubated for 4 h in a deubiquitination buffer in the presence or absence of the recombinant USP25 proteins. Immunoblotting analyses were probed with the indicated antibodies. Data represents n=3 separate experiments. Graphs show mean ± SD and “*” denotes p<0.05.