Arpp19 ablation promotes premature dephosphorylation of key cyclin B/CDK1 substrates and disrupts the temporal order of events of mitotic exit. (A) GFP- or GFP-Cre–treated MEFs were enriched in mitosis by nocodazole and isolated by shake-off. Cells were then released and collected at the indicated time points. Cell lysates were analyzed by Western blotting. Cyclin B1 and securin levels were quantified using ImageJ and are represented relative to the G2 levels. (B and C) Arpp19^Lox/Lox transduced MEFs were fixed and incubated with the indicated antibodies, and images were acquired by confocal microscopy. Arrowheads highlight mislocalization of PRC1 and Aurora B and cytokinesis furrows. Bar, 10 µm. The phenotypes observed in cells from B and C were counted (data from three different experiments). (D) CDK1 activity was tested using histone H1 (H1K) as substrate. Mitosis corresponds to nocodazole-arrested GFP-transduced Arpp19^Lox/Lox MEFs. H1K signals were quantified and corrected to CDK1 levels and are represented as relative to the H1K signal in mitotic cells. Data from two experiments. (E) GFP- or GFP-Cre–expressing adenoviruses were synchronized (Noco) or not (Inter) in mitosis by nocodazole block and incubated or not with OA. Cell lysates were analyzed by Western blotting. Signal intensities were measured by densitometry and normalized and are represented as the mean ± SD of two independent experiments.