Mps1 kinase activity is continuously required to maintain Bub1 and all other SAC effectors at unattached kinetochores. (A and B) Mps1^wt and Mps1^as cells were treated with nocodazole for 4 h to activate the SAC and treated with nocodazole, MG132, and/or 3MB-PP1 for an additional 2 h. Cells were fixed and stained with antibodies against the indicated SAC or kinetochore components (green) and CREST antiserum (red). Note that cells not treated with 3MB-PP1 retained normal kinetochore localization patterns and, thus, have been omitted from these montages for clarity. Additional kinetochore/centromere proteins analyzed in this assay are shown in Fig. S2. (C) Kinetochore (KT)-specific signal intensities were determined in 3MB-PP1–treated Mps1^as and Mps1^wt cells (>100 kinetochores in more than five cells per sample) and normalized to the equivalent values in untreated Mps1^wt cells. Error bars indicate SEM. Bars, 10 µm.