Figure EV2. Destruction assays using mutant APC/Cs (1–2A and 1‐S558A).

1. Cdc20‐dependent destruction assay in anaphase. The purified recombinant WT APC/C or its derivatives (1–2A and 1‐S558A) was incubated with its substrates (³⁵S‐labelled cyclin B and a version of cyclin B lacking the N‐terminal 67 residues, Δ67) in APC/C‐depleted (ΔAPC) interphase extracts supplemented with non‐degradable cyclin B at 23°C. Samples taken at indicated time points were analysed by SDS–PAGE and autoradiography.
2. Quantification of (A). The relative cyclin levels are shown, normalised with reference to the intensities found at time 0 for each time point. Error bars, SEM from three independent experiments.
3. Cdh1‐dependent destruction assay. The purified recombinant WT APC/C or its derivatives (1–2A and 1‐S558A) was incubated with its substrates in APC/C‐depleted (ΔAPC) interphase extracts supplemented with Cdh1 at 23°C. Samples taken at indicated time points were analysed by SDS–PAGE and autoradiography.
4. Quantification of (C). The relative cyclin levels are shown, normalised with reference to the intensities found at time 0 for each time point. Error bars, SEM from three independent experiments.