Mps1 promotes chromosome biorientation independently of aurora B. (A) Cells of the indicated genotypes were treated with 3MB-PP1 and MG132 for 1 h, fixed, and stained to detect centromeres (CREST; green), spindle MTs (α-tubulin; red), and chromosomes (DAPI; blue). (B) Mitotic Mps1^wt and Mps1^as cells were collected via STLC treatment and shake off and maintained in the presence of STLC, MG132, and/or 3MB-PP1 for 2 h. Lysates were resolved by SDS-PAGE and quantitative immunoblotting. The ratio of phosphorylated histone H3 (pH3) to tubulin was computed for each lane and normalized to the ratio in STLC-treated Mps1^wt cells. (C) Cells were treated with nocodazole (noc) for 4 h to activate the SAC and treated for an additional 4 h with nocodazole, MG132, and/or 3MB-PP1. Centromeres (CREST) and serine 7–phosphorylated CENP-A (pCENP-A) were detected by immunofluorescence microscopy. (D) Quantification of results in C is shown. At least 100 centromeres in at least five cells were scored per sample. Error bars indicate SD. (E) MPS1^flox/Δ cells were infected with Adβgal (top) or AdCre (bottom). 3 d later, both populations were treated with STLC and MG132 for 30 min, fixed, and stained with the indicated antibodies. BubR1 loss from kinetochores was used as a functional marker of Mps1 inactivation (Fig. 7). Bars, 10 µm.