Binding of phosphorylated APC/C^Cdc20 to cyclin B1 starts in mitosis. (A) Cyclin B1 IPs from synchronized G2 phase and nocodazole-arrested mitotic cells (mitosis) are shown. See Materials and methods for synchronization details. IPs were analyzed for coprecipitating proteins by Western blotting probed for anti-APC3 (top; showing only phosphorylated APC3) or anti–cyclin B1 (middle). (bottom) The presence of unphosphorylated (lane 1) and phosphorylated (lane 2) APC3 in whole cell extracts (WCE) used for the IPs (10% of input) is shown. (B) APC/C IPs using anti-APC6 antibodies on extracts of G2 phase cells and mitotic cells. (C) Cyclin B1 IPs on extracts from G2 or mitotic cells were used in in vitro ubiquitination assays. Mitotic cells arrested in nocodazole were released but kept in mitosis by the proteasome inhibitor MG132 (NR, MG132). Control, GFP IPs. Purified E1 enzyme, the APC/C-specific E2 enzyme UbcH10, ATP, and HA-ubiquitin (HA-Ub) were added to start the reactions. HA-ubiquitin–conjugated reaction products were probed with anti-HA antibody (for further controls see Fig. S3). Asterisks indicate an aspecific background band in the control GFP IPs. (D) Comparison of mitotic cells arrested in the spindle checkpoint (Noco), released from the spindle checkpoint arrest but kept in mitosis by MG132, or allowed to enter mitosis in the presence of MG132; the latter contain high cyclin A levels (top). Cyclin B1 IPs are shown; negative control, securin (Sec) IP. Western blots were analyzed for coprecipitating proteins with the indicated antibodies. (top) Phosphorylated forms of APC3 in cyclin B1 IPs are shown. (E) Cells were transfected and selected for expression of the indicated cyclin–GFP fusion proteins. Mitotic cells that had entered mitosis in the presence of the proteasome inhibitor MG132 were collected by shake off. GFP IPs were probed for coprecipitation of Cdc20. (F) Cells were synchronized in G2 and mitosis as indicated. Anti–cyclin A IPs, anti–cyclin B1 IPs, and mitotic extracts were blotted for APC3, Cdc20, and Mad2. Comparable polyclonal antibodies were used detecting the N-terminal 430 amino acids of the respective cyclins. Markers are given in kilodaltons.