Figure 2.

BubR1 ABBA²⁷² and ABBA³⁴⁰ Are Essential for the MCC to Bind and Inhibit the APC/C

(A) Apo-APC/C immunoprecipitated from Cdc20-depleted mitotic HeLa cell extracts was incubated with recombinant SBP-Cdc20 and increasing amounts of recombinant MCC (rMCC) containing wild-type, ABBA²⁷² mutant, or ΔD-box mutant of BubR1 (left) or WT or ABBA³⁴⁰ mutant (right). Results shown are representative of three independent experiments.

(B) Ubiquitylation assays shown in (A) were quantified as shown in Figure S1D. Mean ± SD of three independent experiments is shown. Ratios between Cdc20 (MCC) and SBP-Cdc20 added to the reaction are quantified in Figures S1E and S1F.

(C) HeLa FRT/TO cell lines stably expressing inducible siRNA-resistant, FLAG-mRuby (FR)-BubR1 WT or mutants were transfected with siRNA to deplete endogenous BubR1. Anti-FLAG immunoprecipitations from nocodazole-arrested cells were analyzed by immunoblotting and visualized on a LI-COR Odyssey scanner.

(D) Mean ± SEM of protein levels of the experiments shown in (C) from four (left graph) or three (right graph) independent experiments. See also Figure S1.