Cytosolic-specific rescue of Mps1 inhibition restores mitotic timing and SAC proficiency to human cells. (A) Cells were fixed and stained with antibodies to centromere autoantigens (CREST; red) and transgene-encoded Mps1^wt and Mps1^ΔN (green). (B) Mps1^as, Mps1^as/wt, and Mps1^as/ΔN 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 Bub1 (green) and CREST antiserum (red). Kinetochore (KT)-specific signal intensities were determined in 3MB-PP1–treated Mps1^as, Mps1^as/wt, and Mps1^as/ΔN cells (>100 kinetochores in more than five cells per sample) and normalized to values in untreated Mps1^as cells. Bar, 10 µm. (C) Mitotic cells were harvested by overnight STLC treatment and shake off and incubated in medium containing STLC, 3MB-PP1, and/or MG132 for 2 h. Extracts and Cdc20 immunoprecipitates were analyzed as in Fig. 5. Recovery of Mad2 relative to Cdc20 was determined and normalized to its recovery from STLC- and MG132-treated Mps1^as cells (= 1.0). Black lines indicate that intervening lanes have been spliced out. (D) Quantification of mitotic timing is shown. Cumulative frequency of anaphase onset is plotted as a function of time after NEB. (E) Mps1^ΔN rescues the SAC. Cells were traced by phase-contrast microscopy during treatment with nocodazole ± 3MB-PP1 (compare with Fig. 1 F). Error bars indicate SEM.