Fig. 9. Bub1 maintains the BubR1-APC/C interaction following loss of kinetochore–microtubule interactions. Cells were transfected with control or Bub1 siRNA duplexes and then synchronized in mitosis using nocodazole and selective detachment. Following removal of the nocodazole, the cells were then replated for two hours in nocodazole or taxol, plus or minus ZM447439 as indicated. MG132 was also added to maintain the mitotic state. The cells were then reharvested, and protein extracts prepared and analyzed by ion exchange to resolve BubR1-S and BubR1-L. (A) Western blot showing that when Bub1 is repressed, BubR1-L is less abundant in cells released into nocodazole and ZM447439. The blot shown is representative of three independent RNAi experiments. (B) Quantitation of the BubR1-L:BubR1-S ratio confirming that following repression of Bub1, BubR1-L is less abundant in cells exposed to nocodazole and ZM447439. Values represent the mean±s.e.m. derived from three western blots.