The C terminus of Kif18b is a microtubule-binding domain. (A) Top: Schematic diagram representing Kif18b structural domain organization. EB-binding motifs (EBMs) are indicated. Bottom: Amino acid sequence of the C terminus of Kif18b (CT_kif18b). EB-binding motif is highlighted in orange. Red, amino acids that are phosphorylated in vivo and mutated to aspartates in this study; blue, phosphorylatable residues. (B) Representative live-cell images of HeLa cells transfected with full-length and truncated Kif18b motor, costained with Sir-tubulin. (C) Representative images by immunofluorescence of chimeric construct GFP-Kif18a_CT18b (motor domain of Kif18a fused to the C terminus of Kif18b) costained with an anti-EB1 antibody. (D) Representative live-cell images of truncation constructs of Kif18b C terminus. Red arrows indicate Kif18b_487–852 localizing to microtubule (MT) plus ends. Bar, 10 µm. (E and F) Coomassie-stained gel showing recombinantly expressed and purified Kif18b_500–593 (E) and its corresponding elution profile (F; black line, left y axis) from SEC-MALS analysis. Outcome of the MALS analysis for the peak is presented in pink (molecular weight, right y axis). (G) Coomassie-stained gels showing a cosedimentation assay of CT_Kif18b, CT_Kif18bSA, and CT_Kif18bSD in the presence of 2 µM taxol-stabilized microtubules.