Fig. 6.

USP15 isoforms are dynamically expressed and differentially phosphorylated during the cell cycle. a, b A549 cells were synchronised using standard thymidine/nocodazole protocols to enrich for the indicated phases. a USP15 transcript levels remain stable during the cell cycle. Expression of USP15 splice variants and cyclin B1 (CCNB1) were quantified by qRT-PCR, mean data from three independent experiments are expressed relative to actin and normalised to the expression in asynchronous cells (As) for each splice variant. b USP15 protein expression levels oscillate through the cell cycle. USP15 was evaluated by immunoblotting; a representative gel is shown with quantification of total USP15 expression relative to actin below (mean of three independent experiments, error bars SD, one-way ANOVA with Tukey’s multiple comparison test *P ≤ 0.05, **P ≤ 0.01). c–e USP15 isoform-1 increases in abundance by G2/M and becomes phosphorylated. A549 cells were depleted of USP15 isoforms as indicated and protein extracts were compared by immunoblotting for asynchronous cells (As) or cells arrested at G2/M. Separation by 4–12% gradient SDS-PAGE c showing quantification of the siCON2 samples d, and analysis by Phos-tag gel electrophoresis e