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. 2000 Aug;11(8):2821–2831. doi: 10.1091/mbc.11.8.2821

Figure 5.

Figure 5

Polyubiquitination of mammalian E2-C by the APC/C. (A) Recombinant Myc-tagged cyclin B was subjected to an in vitro ubiquitination assay by incubation with rabbit E1, bovine ubiquitin, and either immunopurified human APC/C (hAPC/C) or recombinant mE2-C, as indicated. The reaction mixtures were subjected to immunoprecipitation with anti-Myc, and the resulting precipitates were subjected to immunoblot analysis with antiubiquitin (upper panel) or anti-cyclin B (lower panel). Cyc B, cyclin B. (B) Recombinant mE2-C was subjected to in vitro ubiquitination assay, as in (A), in the absence or presence of purified human APC/C. The reaction mixtures were subjected to immunoprecipitation with anti-mE2-C, and the resulting precipitates were subjected to immunoblot analysis with antiubiquitin (upper panel) or anti-mE2-C (lower panel). (C) Recombinant wild-type (wt) or mutant (C114S) mE2-C was subjected to in vitro ubiquitination assay in the presence of purified human APC/C and was processed as in (B). (D) Recombinant GST fusion protein of mutant (C114S) mE2-C was subjected to in vitro ubiquitination assay in the absence or presence of wild-type mE2-C, together with purified human APC/C and processed as in (B).