a, DAPI staining of the Cdk1cdc2.33 strains in which mitotic progression has been synchronized by transient arrest at the restrictive temperature of 36 °C. While two evenly sized chromatin masses are generated by the anaphase in wild-type cells, chromosome segregation is uneven in the mutant lines and lagging chromosomes are frequently observed. The frequency of phenotypes is presented in Fig. 2e. b, The PP2A-B56Par1 phosphatase assay was carried out as previously described, in which recombinant GST–Rec8391–561 was phosphorylated by a fusion between glutathione and S. pombe casein kinase I (Hhp2) that had also been produced in Escherichia coli22. PP2A-B56Par1.HA was isolated from 2 × 108 cells with 12CA5 antibody under non-denaturing conditions. Phosphatase activity was calculated from the reduction of 32P incorporation in the GST–Rec8391–561 substrate per unit B56Par1.HA. The level obtained in this assay (and every assay presented in this study) was normalized to the reduction in substrate phosphorylation displayed by a B56Par1 precipitate from 2 × 108 cells of an asynchronous B56par1.HA culture run on the same gel (the second lane in b). OA, okadaic acid. c, PP1Dis2 activity assays conducted in parallel with the same samples used in b. These assays established that both the recombinant rabbit PP1γ and the PP1Dis2 samples that were added to the PP2A-B56Par1 assay in a contained PP1 phosphatase activity. We conclude that we have successfully re-established the phosphatase assays described previously22 and that the addition of PP1 to this assay did not alter the phosphorylation status of the phosphorylated GST–Rec8 substrate, indicating that PP1Dis2 displays no activity towards phosphorylated GST–Rec8 used in this PP2A-B56Par1 enzyme assay. d, This panel presents blots of protein levels (left), PP1Dis2 activity assays (centre) and PP2A-B56Par1 phosphatase assays (right) of the PP1Dis2 samples used in the add back experiments in g (bottom blot) and h (bottom three blots). The blot of PP1Dis2 levels on the left shows that similar levels of the different PP1Dis2 proteins were added in each case, while the central panel shows that these samples possessed PP1Dis2 activity. The panel on the right shows that none of the PP1Dis2 samples exhibited any activity in the PP2A-B56Par1 phosphatase assay. In other words, there was no PP2A-B56Par1 in these pull downs despite the fact that PP1Dis2 is able to bind to PP2A-B56Par1. The absence of PP2A-B56Par1 from these samples is either due to the fact that only a minor fraction of the PP1Dis2 complex formed a complex with PP2A-B56Par1, or, as we anticipate, that the high salt (1.2 M NaCl) conditions we used in the immunopreciptation reactions that isolated these PP1Dis2 molecules for the add back experiments (g, h and Fig. 3c, d) had disassociated any PP2A-B56Par1 molecules that partnered these PP1Dis2 molecules in vivo. e–k, PP2A-B56Par1 assays as for b. A quantitative plot of the data in g (top blot) is shown in Fig. 3b while the data from g (bottom blot) and h are represented in the plots in Fig. 3c, d and data from i–k are shown in Fig. 3g–i. For the experiments in g (middle and bottom blots) and h we exploit the redundancy between PP1Dis2 and PP1Sds21 to use PP1Sds21 to provide essential PP1 function and support the viability of PP1dis2.Δ cells2. The PP2A-B56Par1 complexes isolated from these cells have therefore never been exposed to PP1Dis2 regulation and so will be fully phosphorylated on PP1Dis2-target sites. The addition of PP1Dis2 in vitro enables us to assess the impact of dephosphorylation of these sites. To ensure that there could be no cell cycle dependency to any outcome in these assays we assessed the impact of PP1Dis2 addition on PP2A-B56Par1 samples from all stages of a Cdk1cdc2.33 synchronized mitosis. The data from these experiments that are presented in Fig. 3 and in d, g and h clearly demonstrate that the addition of active PP1Dis2 to naive PP2A-B56Par1 complexes that have never been exposed to PP1Dis2 in vivo was able to reactivate the PP2A activity as long as the PP1Dis2-docking site within B56Par1 was intact. In contrast, PP1Dis2 addition failed to reactivate these naive PP2A-B56Par1 complexes when the PP1Dis2-docking site in B56Par1 had been mutated to block PP1Dis2 recruitment, or the Cdk1–cyclin B inhibition site at T316 of PP1Dis2 had been mutated to aspartic acid to mimic the phosphorylated state. Biological replicates: for b, n = 3; for c and d, n = 1; for all other panels, n = 2.