Extended Data Figure 7: Residue 378 within the PP1-docking site of B56^Par1 was dephosphorylated by PP2A-B55^Pab1 that had been activated by PP1^Dis2 recruitment to its own PP1-docking site.

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 B56^Par1.HA fusion protein or commercial rabbit antibodies raised against human B56ε were used to precipitate B56^Par1 and probed as indicated. b, Validation of the PP2A-B55^Pab1 activity assay. This assay exploits the fact that PP2A-B55^Pab1 removes B56-Phos reactivity from B56^Par1.HA. The phosphorylated B56^Par1.HA substrate was precipitated from cells in which mitotic progression had been arrested by incubation of Eg5^cut7.24 cells at the restrictive temperature for 3 h. Eg5^cut7.24 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 B56^Par1 precipitates were free of any B56^Par1 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 B56^Par1.HA substrate. Lane 2 on the right shows that that this procedure disassociated the B56^Par1 subunit from the other subunits of the tripartite PP2A-B56 complex whereas non-denaturing immunoprecipitation conditions (lane 1) do not. The C^Ppa1 and C^Ppa2 catalytic subunits are detected with a commercial antibody. The B56^Par1 samples generated in this way were incubated with the indicated components. ‘Eluted’ indicates reactions in which the B55^Pab1 immunoprecipitate was incubated with a peptide corresponding to the PP1^Dis2-docking site on B56^Par1 (CWPKVNSSKEVLF) to disassociate the PP1^Dis2 enzyme from the PP2A-B55^Pab1 complex before a re-purification isolated the PP1^Dis2 complex once more. It was this re-isolated PP1^Dis2 that was used for the assay in lane 9 while the residual PP2A-B55^Pab1 was used in the assay in lane 6. The impact of this disassociation can be seen by comparing lanes 8 and 9. The PP1^Dis2 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 PP1^Dis2 precipitates. The inhibitor profiles all support the conclusion that this assay specifically detected PP2A-B55^Pab1 activity. Phos-Stop is a commercial pan-phosphatase inhibitor. c–f, PP2A-B55^Pab1 activity assays of cultures in which mitotic progression had been synchronized by the Cdk1^cdc2.33 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.