hSpindly C-terminal is required for KT localization. (A) A schematic diagram of hSpindly depicting truncation mutants (+, KT localization; −, no KT localization). KT localizing capability of each construct was analyzed under vinblastine treatment, which maximally loads checkpoint proteins on KTs. Amino acid numbers are indicated. (B) HeLa cells were transiently transfected with EGFP-hSpindly fusion constructs (shown in A) and KT localization ability of each construct was analyzed using fluorescence microscopy. DAPI stains chromosomes. Representative images show that the C-terminal 294 to 605 aa of hSpindly are required for KT localization. Bar, 10 µm. (C) A schematic diagram depicting the location of the insertion mutants generated in the hSpindly protein. Constructs shown in red were negative for KT localization and constructs shown in blue localized to KTs only under vinblastine treatment. Site of insertion and inserted residues are shown in Table 1. (1D) HeLa cells transiently transfected with EGFP-hSpindly insertion constructs were analyzed for their KT localization ability. Representative images show that the far C terminus is essential for KT localization. Bar, 10 µm.