Figure 3.
The cold stability of KT–MT connections is reduced in cells expressing BubR1T620A. (A) Schematic representation of BubR1 siRNA, rescue transfection, and cold treatment protocol. HeLa S3 cells were simultaneously transfected with the indicated Flag-BubR1 constructs and BubR1-3′ duplexes. After 36 h, cells were treated with Monastrol for 5 h, followed by a 1-h release in the presence of the proteosome inhibitor MG132, resulting in a metaphase-arrested population. Cells were then incubated for the indicated times at 4°C before they were fixed and processed for immunofluorescence microscopy. (B) Cells treated according to the protocol outlined in A were costained for α-tubulin (green), KTs (CREST) (red), and Flag (not shown). Bar, 10 μm. Representative KT–MT connections were enlarged and are shown on the right. (C) Quantification of MT density in B. Average MT intensity (mean ± SE; n ≥ 10 transfected cells) was measured in each condition. Intensities are expressed relative to total cellular areas and normalized against T0 for each condition (MT density at T0 = 100%).