Mps1 continuously stabilizes Cdc20 inhibitory complexes in M phase. (A) Mitotic cells were harvested by STLC treatment and shake off and incubated in medium containing STLC, 3MB-PP1, and/or MG132 for 2 h. Extracts were immunoprecipitated with antibodies to Cdc20 and resolved by SDS-PAGE. Levels of BubR1, APC8, Mad2, and Cdc20 in each IP were determined by quantitative immunoblotting. The ratio of each protein to Cdc20 was computed and normalized to ratios obtained from STLC-treated Mps1^wt cells (= 1.0). (B) Extracts were analyzed as in A except that Mad2 antibodies were used for IP, and Mad1 antibodies were also used for immunoblotting. (C) Cells were arrested in M phase via overnight treatment with nocodazole. At time 0, cells were either treated with 3MB-PP1 or left untreated as a control. Samples were collected at 30-min intervals for analysis of cyclin B levels (left) and determination of mitotic indices (right). Black lines indicate that intervening lanes have been spliced out. Error bars indicate SEM.