The Phe and D boxes of BubR1 contribute to APC/CCdc20 inhibition in vitro in the absence of Mad2.
A, anti-Myc blot of the in vitro ubiquitination reactions of APC/CCdc20 with cyclin B11–97-Myc as the substrate, in the presence of the indicated BubR1-Bub3 proteins at 200 nm. B, schematic drawing of miniBubR1, a miniaturized BubR1 protein containing only four Cdc20-binding motifs: KEN1, KEN2, Phe, and D boxes. C, anti-Myc blot of the in vitro ubiquitination reactions of APC/CCdc20 with cyclin B11–97-Myc as the substrate, in the presence of increasing concentrations of BubR1-Bub3 or miniBubR1. D, anti-Myc blot of the in vitro ubiquitination reactions of APC/CCdc20 with cyclin B11–97-Myc as the substrate, in the presence of the indicated miniBubR1 proteins at 200 nm. E, anti-Myc blot of the in vitro ubiquitination reactions of APC/CCdc20 with cyclin B11–97-Myc as the substrate, in the presence of the indicated BubR1-Bub3 proteins at 85 nm with or without 1.25 μm Mad2 L13A protein.