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Fig. 4. Effect of reduced tension on the kinetochore localization of Mad2, Bub1 and BubR1. (A) Cells were incubated with 10 nM taxol for 10 minutes and then processed for immunofluorescence with antibodies against Mad2, Bub1 or BubR1 (red) and a-tubulin (green); DNA was stained with DAPI (blue). Mad2 and Bub1 are hardly detectable at kinetochores of aligned chromosomes, whereas BubR1 consistently localizes at all kinetochores of the metaphase plate. Mono-oriented chromosomes (arrows) exhibit BubR1 staining at both kinetochores while Mad2 and Bub1 labelling is only observed at unattached kinetochores. Scale bar: 5 µm. (B) Quantification of Mad2, Bub1 and BubR1 immunoreactivity patterns at sister kinetochores of mono-oriented chromosomes: labelling at both kinetochores (+/+), labelling only at unattached kinetochores (+/–) and no labelling at either kinetochore (-/-). Note that, whereas BubR1 labelling at mono-oriented chromosomes is mainly symmetric, Mad2 and Bub1 show staining only at unattached kinetochores.





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