Phospho-mimicking Dsn1 mutation enhances Mis12C binding to CENP-C. (A) HeLa cells expressing Dsn1-WT/EE-GFP were analyzed by time-lapse microscopy. GFP images of representative cells at the indicated times (in minutes) were shown. Metaphase was used as the reference point (time 0). (B) Interphase cells expressing Dsn1-WT/EE-GFP were stained with the indicated antibodies and DAPI. (C–E) HeLa cells expressing Dsn1-WT/EE-GFP were transfected with Ndc80-WT/NLS-mCherry plasmids, and stained with the indicated antibodies and DAPI. (F) HeLa cells expressing Dsn1-EE-GFP transfected with siCENP-C and treated with thymidine were stained with the indicated antibodies and DAPI. (G) In vitro binding assays between recombinant Dsn1-WT/EE-containing Mis12C with or without Ndc80C and ³⁵S-labeled CENP-C (residues 1–71; right). The relative CENP-C binding intensity is indicated at the bottom. Mis12C and Ndc80C were stained with CBB (left). The asterisk indicates a degradation band of Ndc80. (H) Sequence alignment of the basic motif encompassing the two Aurora B phosphorylation sites in Dsn1 proteins from different species (Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Xl, Xenopus laevis). (I) HeLa cells expressing Dsn1-WT/Δ92-113-GFP were analyzed by time-lapse microscopy. (J) Lysates and α-GFP IP of mitotic HeLa cells expressing Dsn1-WT, -AA, -EE, or -Δ92–113-GFP were blotted with the indicated antibodies. Bars: (A and I) 10 µm; (B–F) 5 µm.