a, Validation of the B56-Phos antibody that was raised against and affinity purified with a peptide in which residues corresponding to S377 and S378 were each phosphorylated. Immunoprecipitates in which the 12CA5 monoclonal antibody was used to recognize the HA epitope on a B56Par1.HA fusion protein or commercial rabbit antibodies raised against human B56ε were used to precipitate B56Par1 and probed as indicated. b, Validation of the PP2A-B55Pab1 activity assay. This assay exploits the fact that PP2A-B55Pab1 removes B56-Phos reactivity from B56Par1.HA. The phosphorylated B56Par1.HA substrate was precipitated from cells in which mitotic progression had been arrested by incubation of Eg5cut7.24 cells at the restrictive temperature for 3 h. Eg5cut7.24 is a temperature-sensitive mutation in the kinesin 5 motor protein that is required for interdigitation of the two halves of the bipolar spindle36. To ensure that these B56Par1 precipitates were free of any B56Par1 partners, the total protein content of intact cells was precipitated by trichloroacetic acid (TCA) treatment before cells were homogenized and resuspended in 2% SDS. After a centrifugation clearing step, the addition of 1% Triton X-100 to the resultant supernatant sequestered the SDS into micelles thereby generating conditions for immunoprecipitation (IP) of phosphorylated B56Par1.HA substrate. Lane 2 on the right shows that that this procedure disassociated the B56Par1 subunit from the other subunits of the tripartite PP2A-B56 complex whereas non-denaturing immunoprecipitation conditions (lane 1) do not. The CPpa1 and CPpa2 catalytic subunits are detected with a commercial antibody. The B56Par1 samples generated in this way were incubated with the indicated components. ‘Eluted’ indicates reactions in which the B55Pab1 immunoprecipitate was incubated with a peptide corresponding to the PP1Dis2-docking site on B56Par1 (CWPKVNSSKEVLF) to disassociate the PP1Dis2 enzyme from the PP2A-B55Pab1 complex before a re-purification isolated the PP1Dis2 complex once more. It was this re-isolated PP1Dis2 that was used for the assay in lane 9 while the residual PP2A-B55Pab1 was used in the assay in lane 6. The impact of this disassociation can be seen by comparing lanes 8 and 9. The PP1Dis2 immunoprecipitate exhibited a low level of B56-Phos phosphatase activity when the elution step was not performed. Displacement of partner molecules through peptide incubation removed the activity that reduced B56-Phos reactivity in the uneluted PP1Dis2 precipitates. The inhibitor profiles all support the conclusion that this assay specifically detected PP2A-B55Pab1 activity. Phos-Stop is a commercial pan-phosphatase inhibitor. c–f, PP2A-B55Pab1 activity assays of cultures in which mitotic progression had been synchronized by the Cdk1cdc2.33 arrest/release protocol. The quantification of these assays is presented in Fig. 4f–i. Biological replicates: n = 2 for all panels except the right panel of b, where n = 1.