Chk2 is required for Mps1 localization and stability in nocodazole. (A) Cells expressing Mps1-GFP transfected with negative siRNA (control) or Chk2 siRNA (siChk2) were treated with 3.32 µM nocodazole (nocod) in the presence of AZ3146 (AZ) and MG132 for 4 h. Boxed values show mean Mps1-GFP/Hec1 fluorescence intensity ± SDs. Values in square brackets show kinetochore pairs and number of cells analyzed. Bars, 5 µm. Insets show 1.7× magnification of kinetochores. (B) Western blot analysis of total GFP and actin in cells expressing WT Mps1-GFP and treated with 3.32 µM nocodazole for 8 h. Values at control were taken as 1. (C) Cells expressing WT, T288A Mps1-GFP, or untransfected were treated as in B. (top) Western blot analysis of GFP-associated phosphothreonine (pThr) and Mps1 after GFP immunoprecipitation (IP). (bottom) Western blot (WB) analysis of total GFP and actin. Values at WT were taken as 1. (D) Western blot analysis of total GFP and actin in cells expressing T288A or T288E Mps1-GFP and treated as in B. Values at control T288E were taken as 1. (E) Western blot analysis of total phosphorylated Y15 (pY¹⁵), Cdk1, and actin. Tetracycline-induced CHO WT Aurora B cells expressing WT or T288E Mps1-GFP were treated with 3.32 µM nocodazole in the absence or presence of Chk2 inhibitor II for 8 h followed by shake-off. Values at samples without Chk2 inhibitor were taken as 1. (F and G) Mitotic index analysis. Tetracycline-induced WT, S331A, or S331E Aurora B cells expressing GFP or T288E Mps1-GFP were treated with 3.32 µM nocodazole (and AZ3146; G) in the absence (control) or presence of Chk2 inhibitor II (inh II). Mitotic index shows the percentage of mitotic green cells/total green cells. Error bars show the SD from the means of three independent experiments. ***, P < 0.001.