TTBK2-mediated phosphorylation inactivates KIF2A by inhibiting its binding to MTs. (A) The ability of nonphosphorylated and phosphorylated KIF2A to depolymerize GMCPP-MTs. Increasing concentrations of KIF2A were incubated with GMPCPP-stabilized MTs. The supernatant (S) and precipitation (P) fractions were visualized by Coomassie Brilliant blue staining. Phosphorylation by TTBK2 decreased the MT-depolymerizing activity of KIF2A. The plot shows the quantification of remnant MTs in the precipitation fraction as the mean ± SD of three independent experiments. *, P < 0.05 versus nonphosphorylated KIF2A (Student’s t test). (B) Visualization of GMPCPP-MTs using an antitubulin antibody after brief incubation with KIF2A. Bar, 10 µm. (C) The association between KIF2A and GMPCPP/taxol-stabilized MTs. Increasing concentrations of MTs were incubated with a fixed concentration of KIF2A, followed by ultracentrifugation. Bound (precipitation fraction [Ppt]) and unbound (supernatant fraction [Sup]) KIF2A were visualized by immunoblot analysis. The plot shows the amount of KIF2A bound to MTs relative to the total amount of KIF2A as the mean ± SD of three independent experiments. **, P < 0.01 versus nonphosphorylated KIF2A (Student’s t test).