Fig. 2.

Overexpression of Aur-A accelerates the G₂/M transition. Kinase-dead Aur-A delays mitotic entry and leads to monopolar spindles. (A) Recombinant wild-type or kinase-dead D281A Aur-A (100 ng/μl) or buffer was added to cycling extracts at 50 min. Samples were stained with Hoechst dye 33342. At least 100 nuclei were scored for each condition. (B) Samples in A were blotted with the indicated antibodies. (C) Cycling extracts were supplemented with nuclei and rhodamine-labeled tubulin. Wild-type or D281A Aur-A (100 ng/μl) or buffer was added at 50 min. Samples were stained with Hoechst dye 33342. The percentage of spindle structures was averaged from three independent experiments. At each time point, at least 100 spindle structures were counted. (D) Representative spindle structures are shown. (E) Cycling extracts lacking sperm nuclei were supplemented with recombinant wild-type Aur-A (100 ng/μl) or buffer at 50 min. Samples were blotted as above.