Skip to main content
. Author manuscript; available in PMC: 2016 Jan 15.
Published in final edited form as: Dev Cell. 2015 Feb 9;32(3):358–372. doi: 10.1016/j.devcel.2015.01.003

Figure 5. The ABBA motif of BUBR1 is required to recruit CDC20 to kinetochores and contributes to the strength of the SAC.

Figure 5

(A) HeLa FRT cell lines expressing stable inducible siRNA-resistant wt and mutant BubR1 were transfected with control siRNA or siRNA against BubR1. Immunoblotting analysis shows the expression levels compared to endogenous BubR1 and the efficiency of knockdown. Actin is a loading control.

(B) Taxol-treated HeLa FRT cell lines stably expressing inducible siRNA-resistant Flag-mRuby BubR1, were transfected with the indicated siRNA. Cells were imaged by DIC time-lapse microscopy. Median values of the time spent in mitosis are shown and analysed using an unpaired t-test. (n=3.) See also supplemental Fig. 4A.

(C) RPE1 FRT Venus-CDC20 knock-in cell lines stably expressing inducible siRNA resistant Flag-mRuby BubR1 were treated with control siRNA or siRNA against BubR1 and Taxol added. Images of the first frame after NEBD are shown for each BubR1 protein. Scale bars 10 μm. See also supplemental Figs. 4B & 5.

(D) Box and whisker analysis of kinetochore (KT) localised CDC20 in cells transfected with the indicated siRNA and rescued with wt and mutant BubR1, examples shown in (C). KT signal is relative to the background. Median values of the KT intensity are shown. At least 170 KTs were analysed from at least 11 cells. (n=3.) Unpaired t-test used for statistical analysis.

(E) Levels of KT localised CDC20 and BubR1, relative to the background, are shown for the same cells analysed in (D).