Extended Data Figure 1: Apcin binds Cdc20 and inhibits APC/C-dependent proteolysis in Xenopus extract.
From: Synergistic blockade of mitotic exit by two chemical inhibitors of the APC/C
a, Effects of apcin and derivatives (200 μM) on degradation of a cyclin B1 N-terminal fragment (cycB1-NT) in mitotic Xenopus egg extracts. Substrate was expressed in reticulocyte lysate and labelled with [35S]methionine. Samples were analysed by SDS-gel electrophoresis and phosphorimaging. Quantitation of the 40-min time point from three independent experiments is shown in Fig. 1b. b, Apcin-M and TAME do not inhibit binding of Cdc20 to apcin-A resin. The experiment was performed as shown in Fig. 1e except that the inactive apcin derivative apcin-M or the Cdc20-IR tail antagonist TAME were also tested and [35S]Cdc20 was detected by autoradiography. c, Apcin-A interacts strongly with Cdc20, weakly with Cdh1, and shows little interaction with other WD40-___domain proteins. Proteins were expressed in reticulocyte lysate and labelled with [35S]methionine. Left panel, mean value for the percentage bound on the basis of three experiments (error bars, s.e.m.). Right panel, representative autoradiograph of one of three experiments. b, bound; i, 5% input; c, bound to empty resin (control). d, Apcin inhibits degradation of cycB1-NT in Cdh1-treated interphase extract. The addition of roscovitine (75 μM) is required to inhibit Cdk1 activity that suppresses Cdh1-dependent proteolysis. Note that 100 μM apcin is less effective at stabilizing the substrate in Cdh1-activated interphase extract than in mitotic Xenopus extract (Fig. 3b). e, Apcin binds to endogenous Cdc20 in Xenopus extract as measured by a thermal shift assay33. Extract was incubated with varying concentrations of apcin, heated to 46 °C for 3 minutes, and precipitated proteins removed by centrifugation. The soluble fraction was analysed by SDS–PAGE and western blotting for Cdc20. The left panel shows the percentage of soluble Cdc20 (mean ± s.e.m. from three independent experiments). Western blot from one of three experiments is shown below. For comparison, total Cdc20 from interphase extract (and various dilutions) is shown. Right panel, Coomassie-stained gel of soluble proteins, indicating that there is not an observable non-specific stabilization of proteins induced by apcin addition.
