Usp16 is phosphorylated by CDK1 and Plk1. (A, left) Phosphorylation of GST-tagged Usp16 fragments by CDK1. (Right) Coomassie blue staining. (B) Coomassie blue staining of Usp16 fragment 150–600 phosphorylated by Plk1 and/or CDK1. (C) Immunoblot of Plk1 in cell lysates, Usp16 IP, or mock IP (IgG) complex. Mitosis-arrested HeLa cells were treated with DMSO or RO3306. (D, top) Immunoblot of Plk1 coimmunoprecipitated with GFP-tagged WT or S552A mutant Usp16. (Bottom) Immunoblot of GFP as a negative control. (E) The intensity of immunoblot bands of Plk1 shown in C, which was one representative experiment out of three repeats. (F) The intensity of immunoblot bands of Plk1 shown in D, which was one representative experiment out of three repeats. (G) Immunoblot of Usp16 from asynchronous (AS) or mitosis-arrested HeLa cells treated with either DMSO or BI2536. GAPDH was blotted as a loading control. (H, left) In vitro phosphorylation of Usp16 by Plk1. (Right) Coomassie blue staining. (I) In vitro phosphorylation of GST-tagged Usp16 fragments by Plk1 (left) and protein staining (right). (J) Immunoblot of Usp16, active Plk1 (pT210), and S10 phosphorylated histone H3 (H3pS10) in lysates of HeLa cells harvested at the indicated time points after being released from double-thymidine block. Both cyclin B and actin were blotted on the same membrane.