Chk2 is required for BubR1 and Mad2 localization to kinetochores. (A) Western blot analysis of total phosphorylated Y15 (pY¹⁵), Cdk1, and actin. Cells treated with 3.32 µM nocodazole (nocod) and AZ3146 (AZ) for 8 h in the absence (control) or presence of Chk2 inhibitor II. Values at first lane were taken as 1. (B) Cyclin B fluorescence in cells treated as in A. Values show mean Cyclin B fluorescence intensity ± SDs. (C) Localization of BubR1. Cells transfected with negative siRNA (control) or Chk2 siRNA (siChk2) were treated with 3.32 µM nocodazole, MG132, and AZ3146 for 4 h. (D) Cells expressing Mad2-GFP were treated as in C. (E) Localization of Chk2. Cells were treated with 3.32 µM nocodazole or taxol for 4 h. (F) Cells expressing Chk2-GFP were treated as in E. (G) Localization of phosphorylated Chk2-T383 (pT383). Cells were treated with 3.32 µM nocodazole or taxol for 4 h or transfected with Mps1 siRNA (siMps1) and treated with 3.32 µM nocodazole plus MG132 for 4 h. Boxed values show mean green/red fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. Bars, 5 µm. The insets in C–G show 1.7× magnification of kinetochores.