Figure 4.

Activation of mitotic and interphase APC by purified CDC20 and CDH1. (A and B) CDC20 and CDH1 were purified from baculovirus-infected Sf9 cells and analyzed by SDS-PAGE and Coomassie staining (A) and tested for their ability to stimulate the cyclin B ubiquitination activity of purified mitotic Xenopus APC (B). Extracts from noninfected Sf9 cells were subjected to the same purification scheme, and the resulting fraction (Control) was analyzed in the same way. Samples from the ubiquitination assay were taken at 0, 15, and 30 min. (C–F) Immunopurified Xenopus interphase APC (APCⁱ) and mitotic APC (APC^m) were incubated with increasing amounts (0.01–1000 ng) of purified CDC20 (C and E) and CDH1 (D and F), stringently washed, and subsequently analyzed in cyclin B ubiquitination assays. Samples were taken at 0, 5, 10, and 20 min. The data from the 10-min time point were quantitated and are shown as the percentage of ¹²⁵I-labeled cyclin B that has been ubiquitinated. [¹²⁵I]Cyc B, ¹²⁵I-labeled recombinant cyclin B fragment 13–110.