Figure 4. Cdc20 phosphorylation prevents interphase APC/C binding.

(a,b) Stable HeLa cell lines expressing the indicated YFP-tagged Cdc20 proteins were depleted of endogenous Cdc20 and 8 h after release from a thymidine block YFP–Cdc20 complexes were purified. The samples were analysed by SDS–PAGE and probed for APC1, APC3 and APC7 and this was quantified by LI-COR technology. (c) Bar diagram shows the median and range from two (for APC1 and 7) or three (for APC3) independent experiments. Signals were normalized to Cdc20 and Cdc20 WT. (d,e) As in a,b but YFP-tagged Cdc20 WT cells were treated after 7.5 h for 30 min with Calyculin A and then collected. Representative of two independent experiments. (f) Stable HeLa cell lines expressing the indicated YFP-tagged Cdc20 proteins were synchronized as outlined in Fig. 3a and following release from the last thymidine block followed by time-lapse microscopy. The bar graph shows cells entering mitosis after normalization to Cdc20 WT-expressing cells. Average of two independent experiments (at least 127 cells for each condition) with range indicated by the lines. (g) Stable HeLa cell lines expressing YFP–Cdc20^WT or YFP–Cdc20^Ala were synchronized as outlined in Fig. 3a and after release from the last thymidine block mitotic entry was monitored by time-lapse microscopy. In these experiments, Cdc20 was not depleted but cells instead treated with a control siRNA (luciferase (ctrl)) or a siRNA-targeting APC3. Average of three experiments (at least 312 cells in each condition) is indicated in the bar graph with s.d. indicated by the line. An unpaired t-test without assuming equal s.d. was used for statistical comparison (***P<0.0001; NS, non-significant).