a, The temperature of Eg5cut7.24 B55pab1.HA, Eg5cut7.24 B56par1.HA and Eg5cut7.24 cultures that had been grown overnight to early log phase in supplemented EMM2 medium at 25 °C was increased to 36 °C at t = 0 to inactivate this kinesin 5 and so arrest cell cycle progression in mitosis36. Samples were taken for the phosphatase assays and to monitor the degree of mitotic arrest by scoring the frequency of cells with condensed chromosomes36. Protein phosphatase assays were conducted as described for Extended Data Figs 1b, 6b and 7b. Each activity declined as there was an increase in the frequency of cells in which the inability to form a bipolar spindle has triggered a mitotic arrest due to activation of the spindle assembly checkpoint. b, Two-hybrid assays with the indicated human PP1 isoforms with wild-type and mutant isoforms of the conserved core domain (sequences encoding amino acids 84–400) of human B56γ. c, The mutations introduced in each case. d, Blotting the cell extracts from the two-hybrid clones shown in b with 12CA5 and 9E10 monoclonal antibodies that recognized HA and Myc epitopes within the cassettes harbouring the Gal4 activation and DNA-binding domains, respectively, indicated that equivalent protein expression levels were achieved for each version of the protein. Thus, the failure of the PP1-docking site mutants indicates that the change in the PP1-docking site abolished the affinity between the two molecules. Biological replicates: for a and d, n = 1; for b, n = 3.