a, b, Tethering CDC20 to MAD2 prevents MCC disassembly and release from the APC/C. a, Empty plasmids or plasmids encoding Venus–MAD2 were transfected into HeLa cell lines expressing 3×Flag–GBP–CDC20 and the cells synchronized at prometaphase by thymidine release followed by a nocodazole block. Cells were harvested by mitotic shake off and separated into two cultures after washing once in medium. One culture was harvested immediately (−reversine) and the other resuspended in medium containing 1 µM reversine and 10 µM MG132 (+reversine) for 1 h before harvesting. The APC/C was immunoprecipitated with an anti-APC4 antibody and the immunoprecipitates analysed by quantitative immunoblotting. We note that the APC/C preferred to bind endogenous CDC20 over GBP–CDC20 as the co-activator in vivo (see +reversine lane in control cells) but the MCC M2 did not sequester endogenous CDC20 from the APC/C (see +reversine lane in GBP–CDC20 + Venus–MAD2 cells). b, Mean ± s.e.m. of the relative amounts of the indicated proteins in the APC4 immunoprecipitates calculated from four independent biological experiments. The amount of protein bound to the APC/C in the absence of reversine was set to 1 (red line). c– e, Tethering CDC20 to MAD2 prevents MCC disassembly and release from the APC/C in the absence of endogenous CDC20. c, Plasmids encoding Venus–MAD2 were transfected into HeLa cell lines expressing the indicated CDC20 fusion proteins following siRNA treatment against CDC20 for 48 h. Cells were synchronized at prometaphase then treated with reversine, and anti APC4 and anti-GFP immunoprecipitates were analysed as in a. WT, myc–CDC20; GBP, myc–GBP–CDC20. Note that endogenous CDC20 could not be inhibited through exchange into MCC M2 because a core MCC composed of Venus–MAD2 and untagged CDC20 disassembled. d, HeLa cell lines expressing myc–CDC20 (upper blots) or myc–GBP–CDC20 (lower blots) were transfected with a plasmid encoding Venus–MAD2 followed by siRNA treatment against CDC20 for 48 h. Cells were synchronized at prometaphase and treated with reversine as indicated in a. Total cell extracts were analysed by size exclusion chromatography on a Sepharose 6 column and fractions were analysed by quantitative immunoblotting against the indicated proteins and the relative amounts of Venus–MAD2 plotted in panel e with the sum of Venus–MAD2 intensities set to 1. The migration of APC/C or APC/C-MCC is annotated below panel d. All results are representative of three independent biological replicates.