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. 2016 Feb 25;7:10818. doi: 10.1038/ncomms10818

Figure 6. Cdc20 phosphorylation by Bub1–Plk1 is dispensable for MCC formation.

Figure 6

(a) HeLa Tet-On cells stably expressing Flag-Cdc20 WT or 5A were arrested in mitosis by 5 μM nocodazole. The cell lysates and the anti-Flag immunoprecipitates (IP) of these cells were blotted with the indicated antibodies. The graphs at the bottom show the quantification of the relative BubR1 and Mad2 signals normalized to that of total Cdc20 in the IP (mean±range; n=2 independent experiments). (b) Blots of the input and beads-bound proteins of the binding reactions among the indicated proteins. The graphs at the bottom show the quantification of the relative BubR1 and Mad2 signals normalized to Cdc20. (c) Schematic drawing of the experimental design in d and Supplementary Fig. 4b. APC/C is pre-activated with Cdc20 WT or S92E and then incubated with mini-MCC comprising BubR1N, Mad2 in the closed conformation and Cdc20 (WT or S92E). (d) Anti-Myc blot of the in-vitro ubiquitination reactions of the indicated APC/CCdc20 incubated with the indicated proteins and using securin-Myc as the substrate. The asterisk indicates a nonspecific cross-reacting band.