Figure 6. Mad2 catalytically amplifies formation of the final BubR1-APC/C^Cdc20 inhibitor.

(A) The indicated amounts of Mad2, Bub3, and BubR1^N were incubated with preassembled APC/C^Cdc20 and at various time points assayed for the inhibition of APC/C-mediated ubiquitination of cyclin B_1-102. (B) APC/C^Cdc20 inhibition versus time for the assays denoted in (A). Data represent mean ± SEM (n=3). (C) Schematic of the protocol used to reduce endogenous p31^comet and Mad2 or BubR1 levels by siRNA transfection, followed by inhibition and release with the 26S proteasome inhibitor (with 25 nM Velcade) and microtubule inhibitor nocodazole (100 ng/ml), so as to provide a temporal delay in mitotic exit and allow accumulation of mitotic checkpoint inhibitor(s), respectively. (D) Levels of depletion of p31^comet, Mad2 or BubR1 by siRNA transfection. (E) Duration of mitosis determined by time-lapse microscopy for cells treated as in b. Filming began after removal of Velcade and release into nocodaozle. Cells counted in Set A entered mitosis in the presence of both nocodazole and proteasome inhibition, whereas in Set B only cells that entered mitosis after removal of the proteasome inhibitor were included. More than 65 cells were analyzed for each condition.