Chk2 localizes to kinetochores in unperturbed early prometaphase. (A) Localization of Chk2. (B) Localization of phosphorylated Chk2-T383 (pT383). Boxed values show mean pT383/Hec1 fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. (A and B) Bars, 5 µm. The insets show 1.7× magnification of kinetochores. (C and D) Western blot analysis of total pT383, Chk2 phospho-T68 (pT68), Chk2, and actin. Cells were untreated (un) or treated with taxol, 3.32 µM nocodazole (nocod), or etoposide for 8 h followed by shake-off as indicated. Values at untreated were taken as 1. MI, mitotic index; NS, nonspecific. (E) Chk2 in vitro kinase assay. inh II, inhibitor II. (top) Autoradiography analysis of Aurora B substrates. (bottom) Ponceau staining.