Usp16 interacts with and deubiquitinates Plk1. (A) Immunoblot of Plk1 or IgG mock IP complex from HeLa cell lysates. (B) Immunoblot of Usp16 or IgG mock IP complex from HeLa cell lysates. (C) Immunoblot of cell lysates and GFP IP complex. Cells were transfected with cDNAs coding for either GFP or GFP-Usp16. (D) Mitotic cell lysates were incubated with GST, GST-Plk1-PBD, or GST-Plk1-PBD-2A (H538A/K540A) before being blotted with Usp16. (Bottom) Coomassie blue staining. (E) The localization of Usp16 in interphase and mitotic HeLa cells. (F) Immunostaining shows that Usp16 colocalizes with BubR1 on the kinetochores but not with Crest on the centromere. (G) Ratios of the fluorescence intensity of Usp16 and Crest shown in F from three independent experiments with n = 100–150. Error bars indicate the SEM. ***, P < 0.001. (H) Plk1 and Usp16 colocalize in mitotic cells. (I) Immunoblot of Plk1 precipitated from mitotic HeLa cells with or without the knockdown of KLHL22, CUL3, Usp16, or overexpression of GFP-Usp16. LX, long exposure. Bars, 10 µm.