Extended Data Figure 5 : Characterization of the MCC containing D-box or KEN box 2 mutants of BUBR1.

From: The mitotic checkpoint complex binds a second CDC20 to inhibit active APC/C

Extended Data Figure 5

a, Core rMCC assembled with SBPBUBR1 wild type, or ΔD-box, or ΔKEN2 mutants, was purified as in Extended Data Fig. 1a, b and analysed on a LiCOR Odyssey scanner at 680 nm after SDS–PAGE and Coomassie blue R250 staining. b, The core rMCC mutants prepared in a were assayed as APC/C inhibitors in an in vitro ubiquitylation assay as in Fig. 1d. c, Insect cell extracts expressing CDC20 with SBPBUBR1, either wild type, or ΔD-box or ΔKEN2 mutants, were incubated with streptavidin beads. The proteins retained on the streptavidin beads were analysed by quantitative immunoblotting. Results in panels ac are representative of two independent biological replicates. d, HeLa cells were treated with siRNA against BUBR1 and rescued with 3×Flag–Cerulean–BUBR1, either wild-type or the ΔD-box mutant, and mitosis analysed in 0.116 µM Taxol as in Fig. 3d. The time from NEBD to anaphase (or mitotic exit) was measured and plotted as a box and whisker chart. n, number of analysed cells from two independent biological replicates. e, HeLa cells were treated with siRNA against BUBR1 and rescued with siRNA resistant 3×Flag–Cerulean–BUBR1, either wild type or the ΔD-box mutant, then analysed by immunostaining. Cells were stained with anti-Flag M2 and anti-ACA antibodies, and Hoechst 33342, and representative images of prometaphase cells from two independent biological replicates are shown. Scale bar, 10 µm.