In all parts of this figure, red circles depict anti-Endos, whereas blue squares represent anti-CDKS. (A) Anti-Endos is present during M phase. Xenopus CSF (M phase) extracts were incubated at 22°C. At time t = 0, Ca2+ was added to half of the extract to induce M phase exit; control extract without Ca2+ remained in M phase. At the indicated times, aliquots were assayed for anti-Endos and anti-CDKS as described in ‘Materials and methods’. During M phase, anti-CDKS (light blue squares) is undetectable, whereas anti-Endos (light red circles) is active. As the extracts exit M phase (interphase is achieved within 15–20 min of Ca2+ addition; [Yu et al., 2006; Zhao et al., 2008; Castilho et al., 2009]), anti-CDKS activity (dark blue squares) is strongly induced, while anti-Endos (dark red circles) increases about twofold. (B–E) Drug sensitivities of phosphatase activities. Y-axis values represent the percentage of the phosphatase activity for the given combination of extract and substrate measured in the absence of the inhibitor. Anti-Endos and anti-CDKS have similar sensitivities to okadaic acid and fostriecin, but anti-Endos is substantially more resistant than anti-CDKS to tautomycetin and phosphomimetic Endos S68D. In B and C, green triangles represent dephosphorylation activity against CDK-phosphorylated Histone H3; in C, purple stars are activity against CDK-phosphorylated Histone H1v1.0. In part C, the fostriecin resistant portions of the H3 phosphatase (about 40% of the total) and the H1v1.0 phosphatase (about 80% of the total) likely represent PP1 activity. The HeLa extracts examined in panels B–D were from asynchronous cells, the vast majority of which are in interphase. (F) The specific activities of anti-CDKS and anti-H3 increase upon dilution of the extract, presumably because weakly binding inhibitors are titrated away, but the specific activity of anti-Endos increases at most only marginally upon dilution. The y-axis shows the phosphatase activity on the indicated substrates, normalized to the original volume of undiluted extract. In all panels, n = 1; biological and evolutionary replicates of the experiments in panels B–D are presented in Figure 2 figure supplements 1–5.