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Fig. 8. BubR1 only binds the APC/C when the checkpoint is active. Nocodazole arrested HeLa cells were isolated by selective detachment, washed and then replated in either nocodazole or taxol, plus and minus ZM447439. MG132 was added to arrest the cells in mitosis downstream of the checkpoint. After 2 hours the cells were harvested, soluble proteins extracted and resolved by analytical ion exchange and immunoprecipitations to determine the amount of BubR1 bound to the APC/C. (A) In the absence of ZM447439, BubR1-L is abundant when the checkpoint is activated with either nocodazole or taxol (panels i and ii). However, in the presence of ZM447439, BubR1-L is abundant in nocodazole (panel iii) but not taxol-treated cells (panel iv). [Note that while ZM447439 inhibits BubR1 phosphorylation if added prior to mitotic entry (Ditchfield et al., 2003), BubR1 remains phosphorylated if ZM447439 is added to cells already arrested in mitosis, see supplementary material Fig. S4.] (B) APC7 is present in BubR1 immunoprecipitates from cells released into nocodazole plus ZM447439 (lane 7), but not taxol plus ZM447439 (lane 8).
