Fig. 5. Cdc20 incorporation into the MCC correlates with the strength of the SAC.

(a) Isolation of different complexes and Western blot analysis of the different lysates (n=5 extracts for nocodazole; n=4 extracts for Taxol and n=3 extracts for DMA). Error indicate s.e.m. and are representative of at least three independent experiments. (b) Anti-APC4 or anti-CDC20 immunoprecipitates (IP) from extracts of RPE1 cells treated with nocodazole (n=5 extracts), Taxol (n=4 extracts) or DMA (n=3 extracts) and isolated by mitotic shake-off 12-16 hours later. (c-e), The amount of MCC proteins that co-purified with c) APC4 or d-e) CDC20 were analysed by quantitative immunoblotting, normalised to the level in Taxol-treated cells, and displayed as bar graphs. Error bars indicate s.e.m. and are representative of at least three independent experiments. Note that the CDC20 immunoprecipitates in panel b) (last three lanes to the left) and e) were obtained from APC/C-free extracts (in panel a). (f) Schematic of experiments in panels g) and h). (g) Time-course of the percentage of mitotic cells after 12 hours of drug treatment (automatic detection, n>100 cells per condition). Error bars indicate s.d. and are representative of two independent experiments. (h) Same as g) after the addition of AZ3146 (5 μM) (automatic detection, n>100 cells per condition). Inset is the same as g) after addition of RO3306 (10 μM) (automatic detection, n>100 cells per condition). Error bars indicate s.d. and are representative of two independent experiments. (i), Scatter plot displaying the duration of mitosis of RPE1 Cyclin B1-Venus cells treated with AZ3146 (5μM) in the presence of nocodazole (n=26 cells), Taxol (n=30 cells) or DMA (n=25 cells). Inset represents similar conditions except that cells were treated with siRNA against Mad2 (n=28 cells for nocodazole; n=34 cells for Taxol and n=39 cells for DMA). Representative of at least two independent experiments.