Expression of KIF1A constructs and colocalization with cargo. Related to Figs. 3 and 4. Example images of cells expressing KIF1A constructs in neurons and in COS7 cells or together with cargo in neurons and dimerization analysis on Western blot. (A) Representative images of hippocampal neurons expressing KIF1A deletion constructs (shown in Fig. 3 B) including zooms of the axon, marked by the boxes. TRIM46 staining and the KIF1A channel in the axon are shown below. (B) Quantification of the percentage of NPY vesicles colocalizing with KIF1A during live-imaging experiments (N = 7, n = 15 for KIF1A_FL; N = 4, n = 15 for KIF1A_ΔCC2). (C) Representative images of COS7 cells expressing different KIF1A constructs. (D) Representative images of hippocampal neurons coexpressing KIF1A_ΔCC1 and LAMP1 or Rab3. Blue dotted circles indicate examples of distal tips. Merged image of the boxed region is shown on the right. (E and F) Normal (E) and native (F) SDS-PAGE analysis of GFP-fused KIF1A domains expressed in HEK293T cell lysates. Arrows (F) mark the bands of monomeric and dimeric CC2 domain. (G) Representative images of hippocampal neurons coexpressing KIF1A_ΔCC2-GCN4 and LAMP1 or Rab3. Blue dotted circles indicate examples of distal tips. Merged image of the boxed region is shown on the right. (H) Representative image of a hippocampal neuron expressing KIF1A_ ΔCC2-GCN4 (shown in Fig. 4 H) including zoom of the axon, marked by the box. TRIM46 staining and the KIF1A channel in the axon are shown below. Scale bars, 10 µm (A, C, D, and G) and 20 µm (H).