Skip to main content
. 2015 Jun 2;6:7162. doi: 10.1038/ncomms8162

Figure 5. Defining a region in Bub1 required for BubR1 kinetochore localization.

Figure 5

(a) HeLa cells were treated with a control RNAi oligo (luciferase) or a Bub1 RNAi oligo for 48 h and arrested in mitosis using nocodazole for 2 h. Cells were fixed and stained for CREST and Bub1 or BubR1 separately because both antibodies are mouse monoclonal. (b) The kinetochore levels of BubR1 or Bub1 were measured and normalized to CREST. (c) HeLa cells were depleted of Bub1 by RNAi and complemented with the indicated RNAi-resistant Bub1 constructs for 48 h and arrested in mitosis using nocodazole for 2 h. Cells were fixed and stained for GFP, CREST and BubR1. First the level of Bub1-Venus, based on the GFP signal that fully restored BubR1 kinetochore levels, was determined. This level of GFP was then used for all constructs. (d,e) The kinetochore levels of GFP or BubR1 were determined and normalized to CREST. At least 160 single kinetochores were measured from eight different cells and the mean with standard error of mean is indicated. Scale bar, 5 μm.