Fig. 7.
BubR1 second KEN box is required to inhibit substrate binding to the APC/C. (A) Schematic showing the strategy used to evaluate the ability of recombinant proteins to bind the APC/C in vitro. (B) Immunoblot of an APC/C binding assay, probed with antibodies against the APC/C subunit Apc2 and His (note that all the recombinant proteins are His tagged), showing that whereas the ΔK1 mutant binds poorly under all conditions, Mad2 stimulates binding of BubR1 N370 and ΔK2 to APC/CCdc20. (C,E) Immunoblot of an APC/C substrate binding assay showing that the BubR1 N370 ΔK2 mutant cannot prevent substrate binding when added in combination with Mad2. The assays were performed using CycB N90 (C) or securin (E) as APC/C substrates. (D,F) Bar graph quantifying the experiments in C and E, respectively. Values represent the mean ± s.e.m. of three independent experiments.