Fig. 1: CDC14B and CDK1 regulate phosphorylation of USP9X at serine 2563 in mitosis.

a Co-immunoprecipitation of FLAG-tagged CDC14B with endogenous USP9X from HEK 293T cells that were either left untreated or arrested in mitosis using nocodazole (EV = expression vector). Immunocomplexes and respective WCE were probed with antibodies to the indicated proteins. b Immunoblot analysis of U2OS cells that were either synchronized in G1/S phase using a double thymidine block (left panel), or in mitosis using sequential thymidine and nocodazole treatment (right panel). Cells were then released into the cell cycle and collected at the indicated time points. c Immunoblot analysis of U2OS cells that were treated with siRNA directed against CDC14B and synchronized in mitosis as described above. Mitotic shake-off was performed and samples were analyzed by western blot with the indicated antibodies. d Quantification of n = 3 biologically independent experiments conducted as described in c. Ratio paired t-test was applied with **p = 0.0045. e Immunoblot analysis of mitotic U2OS cells after synchronization with thymidine and nocodazole. Mitotic cells were shaken off and kept in nocodazole-containing medium during treatment with either the CDK1 inhibitor RO-3306 or DMSO for 0.5 h. f In vitro kinase assay with C-terminal truncates of USP9X (aa 2165–2570), either the USP9X wild-type form (USP9X^WT) or a USP9X form with a serine to alanine mutation on position 2563 (USP9X^S2563A), that were purified from Escherichia coli and exposed to recombinant active CDK1-Cyclin B in the presence of radioactive ³²P-ATP (CBB, Coomassie Brilliant Blue), *Cyclin B. g Quantification of two independent experiments conducted as described in f. ³²P signals are normalized to the respective Coomassie signal. Mean is displayed from n = 2 biologically independent experiments. h Enzyme kinetics of USP9X^WT and USP9X^S2563A proteins purified from mitotic HEK 293T cells were measured at different Ubiquitin-AMC concentrations. The resulting values for K_M of USP9X^WT or USP9X^S2563A were 0.9090 or 0.7835 µM, respectively, and for V_max 36.69 or 31.06 µM/min, respectively. The values indicated are from n = 1 experiment. Throughout this figure means and standard deviations as error bars are displayed.