CDK1 and Aurora B are necessary for MPS1 recruitment to unattached kinetochores. (A) HeLa MPS1-GFP cells were arrested for 2.5 h with 20 µM MG132 and then treated with 2 µM ZM447439 (+AURKB-i) or 5 µM flavopiridol (+CDK-i) for 10 min. BUBR1 and kinetochores (CREST) were detected using antibodies, and MPS1 was detected using GFP fluorescence. (B) Kinetochore-associated BUBR1 and MPS1-GFP normalized to the mean of the nocodazole-treated control are plotted; error bars indicate the SEM (≥5 cells and 15 kinetochores measured per cell for three independent experiments). (C) Schematic showing the proposed relationship between CDK1-CCNB1 and PP2A-B55 activities and centromeric Aurora B (CPC Cen) and kinetochore MPS1 (MPS1 KT) localization. The bottom panel shows the role of MKLP2 in transport of the Aurora B CPC from centromeres to the anaphase spindle. (D) MPS1 and Aurora B localization were followed in control (siControl) and MKLP2-depleted (siMKLP2) HeLa MPS1-GFP cells arrested for 2.5 h with 20 µM MG132 and then either fixed immediately (+MG132), treated with 3 µM nocodazole for 5 min (+Noc) or 5 µM flavopiridol for 1 min, followed by addition of 3 µM nocodazole for 5 min (+CDK-i +Noc). Aurora B and kinetochores (CREST) were detected using antibodies, and MPS1 was detected using GFP fluorescence. Aurora B and kinetochores (CREST) were detected using antibodies, and MPS1 was detected using GFP fluorescence. (E) Kinetochore-associated Aurora B and MPS1-GFP normalized to the mean of the nocodazole treated control are plotted; error bars indicate the SEM (≥5 cells and 15 kinetochores measured per cell for three independent experiments). (F) MPS1 and Aurora B localization were followed in siControl and PP2A-B55–depleted (siB55) HeLa MPS1-GFP cells. (G) Kinetochore associated Aurora B and MPS1-GFP normalized to the mean of the nocodazole treated control are plotted, error bars indicate the SEM (≥5 cells and 15 kinetochores measured per cell for three independent experiments).