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. 1998 Jun 1;141(5):1181–1191. doi: 10.1083/jcb.141.5.1181

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Mad2 leaves attached kinetochores. (A–C) Micrograph of a late prometaphase PtK₁ cell with one mono-oriented chromosome. This cell was first lysed and extracted to rinse away cytoplasmic Mad2, and then microtubules were depolymerized. The cell was fixed and immunolabeled with CREST serum (B) and antibodies to Mad2 (C). (A) DIC image. (B) An immunofluorescence micrograph showing that kinetochores labeled normally with CREST serum after the treatment used to depolymerized microtubules after cell lysis. (C) An immunofluorescence micrograph showing Mad2 localization. Mad2 is found at the kinetochore that was unattached before treatment (bottom arrow), but is not present on previously attached kinetochores (top arrow). This demonstrates that Mad2 is not simply masked by microtubules bound to the kinetochore, but is lost after microtubule attachment. Bar, 5 μm.