Aurora B phosphorylates Dsn1 and strengthens the CCAN–KMN interaction. (A) Lysates (Input), α-Mis12C IP (Mis12C), and IgG IP of mitotic HeLa cells treated with nocodazole and MG132 in the presence or absence of ZM447439 were blotted with the indicated antibodies. The asterisk indicates the IgG heavy chain in the IP samples. Dsn1 migrated as multiple bands due to phosphorylation. Mis12 and Nnf1 co-migrated. (B) Mis12C was incubated with Aurora B–INCENP and γ-[³²P]ATP, separated by SDS-PAGE, and analyzed by Coomassie brilliant blue (CBB) staining (left) or with a phosphorimager (right). Mis12 and Nnf1 co-migrated. Nsl1 underwent proteolysis. Myelin basic protein (MBP) was used as a positive control. (C) Mis12C containing Dsn1-WT or Dsn1-EE was incubated with Aurora B–INCENP with (+) or without (–) cold ATP. Samples were separated by SDS-PAGE and blotted with the indicated antibodies. Dsn1-EE was recognized by α-pS100 Dsn1. Black lines indicate that intervening lanes have been spliced out. (D) HeLa cells were arrested in prometaphase with nocodazole and incubated with MG132 alone or with MG132 and ZM447439, and then stained with DAPI (blue in the merge), CREST (red), and α-pS100 Dsn1 (green). The boxed regions were magnified and shown in the rightmost column. (E) Cells stably expressing Dsn1-WT, S100E/S109E (EE), or S100A/S109A (AA)-GFP were transfected with siDsn1, treated as in D, and stained with the indicated antibodies. Boxed regions in merged images were magnified and shown in the rightmost column. The normalized kinetochore intensities (mean ± SD, n = 400) of Dsn1-GFP and Knl1of cells were quantified and shown. Bars, 5 µm (1 µm for magnified images).