Extended Data Figure 2: Apcin acts as a competitive inhibitor of APC/C-dependent ubiquitylation.
From: Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C
a, Apcin inhibits formation of high-molecular-mass ubiquitin conjugates of full-length cyclin B1. Mitotic Xenopus extract was pre-treated with the deubiquitinating enzyme inhibitor ubiquitin vinyl sulfone (UbVS, 20 μM) and proteasome inhibitor MG262 (150 μM) to stabilize ubiquitin conjugates. [35S]cyclin B1 was added together with ubiquitin (44 μM) and samples analysed by SDS–PAGE and phosphorimaging. Right panel shows quantitation of the experiment. b, Apcin acts as a competitive inhibitor of APC/C-dependent ubiquitylation. APC/C was purified from mitotic Xenopus extracts and the initial rates of ubiquitylation of HA-tagged cycB1-NT were measured in the presence of methylated ubiquitin to prevent ubiquitin chain elongation. The reaction was stopped at 45 s and the products were detected by anti-HA blot. The left panel shows the anti-HA blot from one experiment; substrate concentrations were 62.5, 125, 250, 500 and 1,000 nM (left to right). Asterisk indicates an SDS-resistant aggregated form of substrate. Quantitation of three independent experiments, and a summary of kinetic parameters, are shown. Note that the effects of apcin in this reconstituted assay performed under initial rate conditions appear distinct from those obtained in crude Xenopus extract. See Supplementary Discussion for a more detailed discussion of these differences.
