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Fig. 4. Aurora-A activation induced by PP2A removal is dependent on Cdc2. Prophase extracts were incubated in the presence of 40 µg/ml of p21^Cip1 and then incubated (C) or not (A) with microcystin-agarose beads at 4°C. Control extracts were obtained by incubation with control beads at 4°C (B). After centrifugation, the supernatants were then incubated in the absence or in the presence of an ATP-regenerating system (ATP) with or without okadaic acid (OA) for 2 hours at 30°C. Samples were collected at indicated times either for histone H1 and H3 kinase assays or for western blotting. A pull-down assay on p13 beads was used to measure Cdc2 kinase activity using histone H1 as substrate. Immunoprecipitates were performed with the anti-Aurora-A antibody and assayed for Aurora-A kinase activity using histone H3-peptide as substrate. Cdc2 and Aurora-A kinase activities are expressed in cpm incorporated in the substrate. Western blots were probed with the anti-Cyclin B2 antibody, the anti-Aurora-A antibody, the anti-PP1 antibody, and the anti-PP2A antibodies as indicated.

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