Figure 6.
Timing of NDC80 phosphorylation during mitosis and mitotic exit. (A) Parental and PPP6C KO HeLa cells stained for Aurora A, the Aurora A activating protein TPX2, active Aurora A pT288, and DNA. Examples of metaphase and anaphase cells are shown. Arrowheads indicate the spread of active Aurora A on metaphase and anaphase spindles in PPP6C KO cells. Note the enlarged metaphase plate in PPP6C KO cells. (B) Synchronized parental and PPP6C KO HeLa cells in mitosis were treated with CDK inhibitor to promote entry into anaphase and mitotic exit. NDC80 phosphorylation was followed using NDC80 pS55 and Phos-tag gels. Mitotic exit was confirmed by blotting for cyclin B, the inhibitory pT320 modification of PP1, and active Aurora A pT288. (C) Parental and PPP6C KO HeLa cells stained for NDC80 pS55 and DNA. Examples of prometaphase, metaphase, and anaphase cells are shown corresponding to the conditions in B. Source data are available for this figure: SourceData F6.