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. 2011 Nov 15;22(22):4236–4246. doi: 10.1091/mbc.E11-03-0216

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© 2011 Hagan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 3: — p31^comet localizes to unattached kinetochores in a Mad2-dependent manner. (A) p31^comet localizes to unattached kinetochores. HeLa cells were stained with DAPI, anti-p31^comet antibodies, and CREST serum. Scale bar, 5 μm. (B) p31^comet binds to kinetochores in prometaphase and requires Mad2. HeLa cells treated with the indicated siRNA were stained with DAPI, CREST, and anti-p31^comet antiserum. Scale bar, 2.5 μm. Indicated sister kinetochore pairs are magnified at right; scale bar, 0.5 μm. (C) Quantitation of p31^comet loss from kinetochores. p31^comet/CREST signal ratios from individual kinetochores are plotted as dots; bars represent mean ratio values. (D) Images captured from a time-lapse sequence showing p31^comet–EYFP localization during mitosis. Kinetochore localization (arrows) is most apparent in early prometaphase when chromosomes are unattached. Upon attachment a portion of the fluorescence accumulates at spindle poles (arrowheads) and in the spindle region (triangles). After all chromosomes no longer have kinetochore staining, anaphase onset begins (dashed lines mark chromosome masses). Time points are in minutes. Scale bar, 10 μm.