Fig. 6.

BubR1 KEN boxes are important for APC/C inhibition. (A) Ubiquitylation assay showing that, in the presence of Mad2, the BubR1 ΔK1 mutant cannot inhibit APC/C^Cdc20. (B) Schematic showing the N370 fragment which encompasses the TPR-like domain and both KEN boxes; plus a Coomassie Blue gel showing purified BubR1 N370 proteins (arrow). The asterisk denotes a contaminant band. (C) Ubiquitylation assay showing inhibition of APC/C^Cdc20 with either BubR1 FL or N370, in the presence of Mad2. (D,E) Ubiquitylation assays showing that in the presence of Mad2, BubR1 N370 ΔK1 does not inhibit APC/C^Cdc20 whereas the ΔK2 mutant only elicits a partial inhibition. The assays were performed using CycB N90 (D) or securin (E) as APC/C substrates. (F) Bar graph quantifying the experiments in A, D and E. In each case, values were normalised to the activity of APC/C in the presence of only Cdc20 (100%) and represent the mean ± s.e.m. of at least two independent experiments each.