CENP-T contributes to KMN kinetochore targeting independently of its NBM. (A) HeLa cells were mock transfected or transfected with the indicated siRNAs, treated with thymidine for 14 h, released into nocodazole-containing medium for 12 h, and treated with ZM for 2 h. The mitotic indices of these cells (mean ± SD [error bars], n = 3) were quantified with FACS. (B) Lysates of cells in A were blotted with the indicated antibodies. (C) HeLa cells were mock transfected or transfected with siCENP-T and arrested in mitosis by nocodazole. Mitotic cells were collected by shake-off. Each sample was divided into two fresh wells. One well was incubated with MG132 (MG) for 2 h (NM) while the other well was treated with both MG and ZM for 2 h (NM+Z). Cells were stained with the indicated antibodies and DAPI. The boxed regions of the merged images of the selected channels were magnified and shown in the rightmost column. The relative kinetochore intensities (mean ± SD, n = 400) of CENP-C and Mis12C were quantified and shown. Bars, 5 µm (1 µm for magnified images). (D) Lysates (Input), α-Mis12C IP (Mis12C), and IgG IP of mitotic HeLa cells transfected with the indicated siRNAs and treated with nocodazole were blotted with the indicated antibodies. (E) In vitro binding assay between recombinant Mis12C and Spc24-25 in the presence of increasing concentrations of a synthetic peptide containing residues 80–104 of human CENP-T with phospho-T85. The reaction mixtures were blotted with anti-Mis12C and anti-Spc24/25 antibodies. (F) Lysates (Input) and α-Mis12C IP (Mis12C) of mitotic HeLa cells transfected with the indicated plasmids and siCENP-C and treated with nocodazole were blotted with the indicated antibodies. Residues 85–99 were deleted in CENP-T ΔNBM. Lines indicate that intervening lanes have been spliced out. (G) Quantification of mitotic indices of HeLa cells expressing indicated proteins that were transfected with siCENP-T and treated with nocodazole and ZM (mean ± SD [error bars], n = 3).