HAP1 contains a novel conserved p150^Glued binding site. (A and B) Example immunoblot and quantification of p150 coimmunoprecipitation by HAP1 or BICD2N (coexpressed in COS-7 cells) and vice versa. n = 3–4 repeats; results were normalized to HaloTag only (negative control; dotted black line); one-way ANOVA of the three conditions with Tukey’s multiple comparisons test (left: BICD2N vs. WT, P = 0.0349; BICD2N vs. TA, P = 0.0114; WT vs. TA, P = 0.7518; right: BICD2N vs. WT, P = 0.0034; BICD2N vs. TA, P = 0.0059; WT vs. TA, P = 0.8347). (C) Schematic showing sequence alignment for the Glued binding motif. Stars indicate point mutant. (D–G) Immunoblotting and quantification show HAP1 (D and E) and Hook1 (F and G) Glued motif mutants (EEAA) coimmunoprecipitation of FLAG-p150 (coexpressed in COS-7 cells). n = 4 repeats; results were normalized to HaloTag only (negative control; dotted black line); paired t test (HAP1, P = 0.0268; Hook1, P = 0.0037). (H and I) Example kymographs and quantification of HAP1+ puncta motile behavior in the mid-axon. n = 13–15 neurons; two-way ANOVA with Bonferroni’s multiple comparisons test (Retrograde WT vs. EEAA, P = 0.0019; stationary/bidirectional WT vs. EEAA, P = 0.0103). Bars throughout show mean ± SEM. *, P < 0.05; **, P < 0.01.