Supplementary Figure 2

(A) Conservation of APC1 residues in different species. (B) Purified GST or indicated GST-Arpp19 proteins were phosphorylated with purified MASTL in the presence of [γ-³²P] labeled ATP and reactions separated by SDS-PAGE and analyzed by autoradiography. GST-Arpp19 proteins phosphorylated with MASTL using ATPγS was incubated with a mitotic extract from HeLa cells expressing myc-tagged PP2AC. Next, the GST-tagged proteins were purified and analyzed for bound PP2A-C. The experiment was performed once. (C) B55 isoforms were depleted as indicated and Cdc20 immunopurified following release from a nocodazole block at the indicated times. The level of Cdc20 T70 phosphorylation and total Cdc20 purified was determined (data represent 1 out 3 experiments with similar results). Quantification of Cdc20 T70 phosphorylation for the specific experiment is shown below. (D) Analysis of the experiments in Fig. 2a measuring the time from nuclear envelope breakdown (NEBD) to metaphase and the time from metaphase to anaphase. The timing of mitotic events was determined from the DIC images and only cells allowing clear determination of metaphase were analyzed. (n = 30 cells analyzed per condition from 3 independent experiments, median with interquartile range indicated by red lines). (E) Analysis of the localization of YFP tagged Cdc20 wt and Cdc20 3SP from cells progressing through an unperturbed mitosis. Representative images from a cell in prometaphase and metaphase are shown. More than 20 cells were analyzed. Scale bars; 10 μm.