Extended Data Figure 4. Effects of apcin on Cdc20 binding to APC/C and stability of APC/C substrates in mitotic Xenopus extract.
a, Example western blot for data shown in Fig. 3a. b, Substrate-mediated recruitment of Cdc20 to APC/C in Xenopus extract is dependent on the D-box motif. Increasing concentrations of wild-type or different D-box mutants of cycB1-NT were introduced into mitotically-arrested Xenopus extract and the APC/C was isolated with anti-Cdc27 antibodies by immunoprecipitation for 1 hour at 4°C. The immunoprecipitate was separated by SDS-PAGE and analyzed by western blotting against Cdc20 and Cdc27. Levels of Cdc20 were quantitated using Image J and normalized to APC/C subunit Cdc27. c, Analysis of effects of apcin and TAME on APC/C substrate degradation in mitotic Xenopus extract. Levels of 35S-labeled substrates were assessed by SDS-PAGE and phosphorimaging. Asterisk represents a non specific band. Images show one of three experiments quantitated in Fig. 3b. d, Experiment performed as in c, but examining combined effects of apcin and TAME. Image shows one of three experiments quantitated in Fig. 3c.