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

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Copyright © 2015 SIBS, CAS

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APC^CDC20 destructs USP22 protein during cell exit from M phase. (a) HCT116 cells were arrested at the G1/S boundary with a double thymidine treatment and then released into fresh medium. Cells were collected every 2 h and lysates were analyzed by immunoblotting with indicated antibodies. (b) HCT116 cells were synchronized in prometaphase with thymidine and nocodazole, then released into fresh medium without or with 20 μm MG132 treatment for the indicated durations. (c) USP22 specifically interacts with CDC20 but not with FBW7 or CDH1. HCT116 cells were transfected with indicated plasmids and USP22 interactions with each of them were analyzed as in Supplementary Figure S1B. (d) CDH1, CDC20 or FBW7 expression plasmids were transfected into HCT116 cells. The endogenous levels of USP22 protein in transfected cells were analyzed by immunoblotting (top panel). (e) CDC20 is required for USP22 destruction in mitosis. Cdc20 was depleted from HCT116 cells using siRNA, cells were synchronized by treatment of nocodazole for 12 h, then mitotic cells were collected by the 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 a loading control. (f) USP22 interacts with APC8. USP22 plasmids were co-transfected without or with each of the indicated APC proteins. Their interactions were determined as described in c. (g) HCT116 cells were transfected with control or CDC20-specific siRNA. The interaction of USP22 with APC8 was examined as in c. (h) APC8 is required for USP22 destruction in mitosis. APC8 was depleted from HCT116 cells using siRNA, then cells were synchronized and analyzed as in e. (i) Sequence alignment of putative USP22 D-box motif compared with those from cyclin A, cyclin B1 and securin. The two residues in USP22 mutated to alanine to yield the USP22 D-box mutant (R98A/L101A) are indicated (upper panel). The conserved D-box sequence of USP22 is indicated (lower panel). (j) The USP22 D-box mutant is stable in cells arrested in mitosis. HCT116 cells stably expressing either wild-type USP22 (WT) or the D-box mutant were synchronized and analyzed as in e. APC, anaphase-promoting complex; siRNA, small interfering RNA; USP2, ubiquitin-specific protease 22.