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. 2015 Oct 13;1:15028. doi: 10.1038/celldisc.2015.28

Figure 3.

Figure 3

USP22 deubiquitinates and stabilizes CCNB1. (a and b) The mRNA levels of USP22, CCNB1, CCNA and CCNE in transfected HCT116 cells (a) or MEF cells (b) were analyzed by real-time PCR. Error bars represent data from three independent experiments. (c) Flag-CCNB1 and HA-Ub expression plasmids were co-transfected with empty vector, myc-USP22 or myc-USP10 into HCT116 cells. CCNB1 ubiquitination was determined by immunoprecipitation of CCNB1 with anti-Flag antibodies and immunoblotting with anti-HA antibody. (d) Deubiquitination of CCNB1 by USP22 in vitro. Ubiquitinated CCNB1 was purified from HCT116 cells transiently transfected with Flag-CCNB1 and HA-Ub expression plasmids, and the cell lysates were incubated with indicated GST or GST-fusion proteins at 37 °C for 2 h and then subjected to SDS–PAGE analysis. CCNB1 ubiquitination levels were determined by immunoblotting with anti-HA antibody. (e) Knockdown of USP22 by siRNA in HCT116 cells promotes CCNB1 ubiquitination. HCT116 cells were transfected with indicated siRNA and ubiquitination assay was analyzed as in c. (f) HCT116 cells stably expressing USP22, its C185A mutant, or USP22 shRNA were synchronized by treatment of nocodazole for 12 h, then mitotic cells were collected by shake-off approach and released into fresh medium for the indicated times. Levels of indicated proteins were detected by corresponding antibodies. Tubulin was used as loading control. (g) The band densities of CCNB1 in f were analyzed using a Bio-Rad imaging software. (h) The mRNA levels of USP22, CCNB1, CCNA and CCNE in the established HCT116 stable cell lines were analyzed as in a). (i) Loss of USP22 facilitates CCNB1 degradation in MEF cells. The indicated proteins in wild-type or USP22 knockout MEF cells were analyzed as in f. (j) HCT116 cells stably expressing control or USP22-specific shRNA were treated with or without MG132 (20 μm) for 2 h before harvested. The expression levels of CCNB1, USP22 and tubulin were analyzed by immunoblotting. CCNA, cyclin A; CCNB1, cyclin B1; CCNE, cyclin E; MEF, mouse embryonic fibroblast; shRNA, short hairpin RNA; siRNA, small interfering RNA; USP2, ubiquitin-specific protease 22.