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Fig. 7. PP1 inhibition is not sufficient to trigger Aurora-A phosphorylation and activation. Prophase extracts (Pro) were depleted in PP2A by incubation with microcystin-agarose beads in the presence or in the absence of p21Cip1. After centrifugation, the extracts were incubated for 2 hours in the presence or in the absence of 500 nM P-DARPP-32 at 30°C plus or minus an ATP-regenerating system (ATP). Samples were collected for kinase assays and western blots. A pull-down assay on p13 beads was used to measure Cdc2 kinase activity using histone H1 as substrate. Immunoprecipitates were performed with 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-P-Aurora-A antibody, the DARPP-32 antibody, the anti-P-DARPP-32 antibody, the PP1 antibody and the PP2A antibodies, as indicated.





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