Fig. 4.

BubR1 KEN boxes are required for SAC function. (A) Immunoblots of HeLa cells showing induction of the Myc-tagged BubR1 KEN mutants following RNAi-mediated repression of the endogenous protein. (B) Immunoblots of Myc immune complexes isolated from HeLa cells arrested in mitosis with nocodazole for 16 hours, probed with antibodies against Myc, Bub3, Cdc20, Mad2 and the APC/C subunit Cdc27. (C) A cumulative plot showing the time arrested in mitosis. At least 50 cells were analysed for each curve. (D) Box and whisker plot showing the amount of time cells take from NEBD to anaphase onset. At least 40 cells were analysed in each case. (E,F) Graphs measuring securin–DsRed fluorescence with maximum fluorescence intensity was normalised to 100%. The data in E and F are the same, but normalised such that in E, T=0 represents NEBD whereas in F, T=0 represents metaphase. Note that when BubR1 function is not rescued, anaphase occurs without obvious metaphase, so only the control, WT and ΔK2 are shown in F. Data are plotted as mean ± s.e.m. of three to five cells in each case.