Mutating Kvβ2 N-terminal phosphosites impacts Kvβ2–EB1 interaction. (A) COS-1 cells cotransfected with EB1-EGFP and WT Kvβ2, or Kvβ2 S31A (ratio 1:1). After methanol fixation, cells were immunostained with anti-Kvβ2 mAb K25/73 (red). Bar, 20 µm. (B) MT recruitment was quantified by dividing the number of cells with MT-like Kvβ2 immunostaining by the total number of cells coexpressing Kvβ2 and EB1; 500 cells were counted from three independent experiments. **, P < 0.01; ***, P < 0.001. (C) GST pull-down assays. Input and products of reactions performed with GST-EB1c on lysates from HEK293 cells expressing WT Kvβ2, or Kvβ2 mutants (S9A, S20A, or S31A) and immunoblotted with an anti-Kvβ2 mAb K25/73.