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. 2020 Jan 1;10(3):1046–1059. doi: 10.7150/thno.36503

Figure 4.

Figure 4

USP28 directly counteracts KLHL2-mediated UCK1 ubiquitination and proteasomal degradation. (A) Screening the deubiquitinating enzymes of UCK1. The constructs as indicated were transfected into HEK293T cells. Then the UCK1-Luc firefly luciferase activity was measured. (B) In HL-60 cells, Flag vector, Flag-USP28-WT or Flag-USP28-CA (C171A), were expressed and treated with CHX (50 μg/ ml) for indicated time intervals. Then the protein levels were analyzed by western blotting (left panel) and statistical analysis of UCK1 was presented (right panel). (C) MV4-11 cells were transfected with control shRNA, USP28 shRNA-#1, USP28 shRNA-#2, or USP25 shRNA, and then treated with CHX. Western blotting was performed and statistical analysis of UCK1 level was presented (left panel). The graph represents mean ± SD. Western blots were also used to examine the protein levels of USP28 and USP25 (right panel). (D) HA-UCK1 and His-ubiquitin were co-expressed with WT-USP28 or catalytic-dead USP28 (C171A) in HEK293T cells. After MG132 (10 μM) treatment for 6 h, Ni-NTA agarose beads were used to pull down the ubiquitinated proteins under denaturing conditions, and the ubiquitination of UCK1 was detected by western blotting. (E) His-USP28 protein and HA-UCK1 were subjected to in vitro deubiquitination. (F) MV4-11 cells stably expressing control or USP28 shRNA-#1, shRNA-#2, were treated with MG132 for 6 h. UCK1 was immunoprecipitated with an anti-UCK1 antibody. Immunoblotting was performed with indicated antibodies. (G) KLHL2 binds to USP28. HEK293T cells were transfected with indicated plasmids. Cell lysates were subjected to immunoprecipitation with anti-KLHL2 antibody and immunoblotting analysis. (H) USP28 inhibited KLHL2-mediated UCK1 ubiquitination. HEK293T cells were transfected with indicated plasmids as indicated and immunoblotting was carried out. (I) KLHL2-mediated proteasomal degradation of UCK1 was suppressed by USP28. HL-60 cells were transfected with indicated plasmids. 24 hours later, cells were cultured in the presence of CHX for the indicated time. Then immunoblotting analysis was performed.