Mps1 accumulates on kinetochores when inhibited. (A) Immunolocalization of LAP-Mps1 in U2OS cells cotransfected with Mps1 shRNA and LAP-Mps1-WT or LAP-Mps1-KD and treated with nocodazole and MG132. Immunoblot shows expression of LAP-Mps1-WT and -KD in whole-cell lysates. (B) Immunolocalization of Mps1 and Mad2 in HeLa cells treated as indicated. Graph represents quantitation of fluorescence intensities (±SEM, 5 cells per condition, 22 kinetochores/cell). (C and D) Quantitation of fluorescence intensities at kinetochores of U2OS cells transfected and treated as indicated (±SEM, 8 cells per condition, 22 kinetochores/cell). (E) UTRM-LAP-Mps1^M602G cells were treated as indicated, and 0.81-µm² areas around single kinetochores (green squares in top panel) or in the cytoplasm (white squares) were bleached at t = 1 s. Graphs show average fluorescence intensities, shaded areas indicate SDs, and percentages indicate average recovery between 10 and 12 s. (F) Mps1 localization on bioriented and mono-oriented kinetochores in a HeLa cell treated as indicated.