Retention and destruction of cyclin B1 critically depends on APC3. (A) Cells were arrested in mitosis by nocodazole after treatment with control siRNA (lanes 1 and 2) or arrested by treatment of siRNA pools targeting APC3 (lanes 3 and 4) or APC2 (lanes 5 and 6). 50% of these mitotic cells were released out of mitosis by the Cdk1 inhibitor roscovitine (Rosco; lanes 2, 4, and 6). Extracts were blotted and probed with antibodies against the indicated proteins. (B) IPs with anti–cyclin B1 antibodies on extracts of mitotic cells arrested in the spindle checkpoint (nocodazole) by allowing the cells to enter mitosis in the presence of MG132 or by treatment of siRNA pools targeting APC3 or APC2. Extracts and IPs were probed with antibodies against the indicated proteins. (C) IPs with cyclin B1 antibodies on extracts of mitotic cells arrested by the spindle checkpoint and treated with control siRNA or an siRNA pool targeting Cdc20. IPs were blotted and probed with antibodies against the indicated proteins. Identical results were obtained by using shRNA to target APC3 (Fig. S5). Markers are given in kilodaltons. WCE, whole cell extract.