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. 1999 Sep 28;96(20):11317–11322. doi: 10.1073/pnas.96.20.11317

Figure 5.

Figure 5

Analysis of the D box dependence of brain APC. (A) Flow cytometric analysis of HeLa cells: HeLa cells were enriched in G1 phase by growth to confluency or arrested in mitosis by treatment with nocodazole, and their DNA contents was analyzed by flow cytometry. (B) Analysis of CDC20 and CDH1 association with APC: APC immunoprecipitates from extracts of confluent and mitotic HeLa cells and from cow brain Q10 fraction were analyzed by immunoblotting with CDC20, CDH1, APC10/DOC1, and CDC27 antibodies. Immunoblots marked with * (CDH1 and APC10) were performed by using an iodinated secondary antibody. (C) Relative ratio of CDH1/APC10 by using the data of the immunoblot shown in B, quantitated by PhosphorImaging. (D) Kinetics of ubiquitination reactions: ubiquitination assays by using immunoprecipitated APC from cow brain, confluent and mitotic HeLa cells were performed with cyclin B 13–110 wild type (wt) or 13–110 Db-AA mutant as described for Fig. 4C. The percentage of cyclin B-ubiquitin conjugates was quantitated for each time point. (E) Ratios of ubiquitination activities toward cyclin B Db-AA vs. activities toward wt cyclin B, calculated for the time point at 10 min.