Either H3pT3 or H2ApT120 alone is sufficient to recruit Aurora B. (A–C) U2OS-LacO cells expressing EGFP-LacI, EGFP-LacI-Haspin-K, or EGFP-LacI-Bub1-K (WT or kinase-dead) were fixed and then subjected to immunofluorescence staining with DAPI and antibodies for H3pT3 and H2ApT120. Example images are shown (A). The relative enrichment of H3pT3 (B) and H2ApT120 (C) at the LacO repeats was quantified in 50 cells. (D and E) U2OS-LacO cells expressing WT or the kinase-dead mutant of EGFP-LacI-Haspin were fixed for immunostaining. Example images are shown (D). The relative enrichment of Aurora B at the LacO repeats was quantified in 50 cells (E). (F and G) U2OS-LacO cells expressing EGFP-LacI or EGFP-LacI-Bub1-K (WT or kinase-dead) were fixed for immunostaining. Example images are shown (F). The relative enrichment of Sgo1 at the LacO repeats was quantified in 50 cells (G). (H) U2OS-LacO cells expressing EGFP-LacI-Bub1-K (WT or kinase-dead) were exposed to nocodazole for 3 h. Mitotic cells were cytospun onto coverslips, fixed, and subjected to immunofluorescence staining with DAPI, ACA, and the antibody for Aurora B. Example images are shown. (I) U2OS-LacO cells expressing the indicated EGFP-LacI fusion proteins were exposed to nocodazole and 5-ITu for 3 h. Mitotic cells were cytospun onto coverslips and fixed for immunostaining as in H. (J) U2OS-LacO cells expressing the indicated EGFP-LacI fusion proteins and transfected with control siRNA or Sgo1 siRNA were exposed to nocodazole for 3 h. Mitotic cells were cytospun onto coverslips and fixed for immunostaining as in H. Means and SDs are shown (B, C, E, and G; unpaired t test). Arrows point to the LacO array. Scale bars, 10 µm.