Decreased surface expression of IGF1R in Kif1b^−/− mouse neuronal axons. (A and B) Surface immunocytochemistry of Kif1b^+/+ and Kif1b^−/− mouse hippocampal neurons at DIV2 against IGF1Rα accompanied by the respective differential interference contrast (DIC) images taken using a Plan Apochromat 20×/0.8 objective (A) and quantification (B). Cells were plated at 8.6 × 10⁴ cells/cm². Note the significant decrease in the surface IGF1Rα staining both in the cell body and neurites. Bar, 10 µm. n = 31–42. *, P < 0.05; ***, P < 0.001, one-sided unpaired Welch’s t test. (C and D) Axonal immunocytochemistry against IGF1Rα and βIII-tubulin of Kif1b^+/+ and Kif1b^−/− mouse hippocampal neurons at DIV7 using microfluidic chamber culture (C) and the quantification (D). (C) Top: Schematic view of the device. Bottom: Immunofluorescent images with the cell body on the left. Note that axonal IGF1Rα but not βIII-tubulin of Kif1b^−/− neurons was significantly decreased compared with that of Kif1b^+/+ neurons. Bar, 5 µm. n = 17–26. ***, P < 0.001, one-sided unpaired Welch’s t test. (E and F) The surface biotinylation assay of primary cultured mouse cortical neurons of the indicated genotypes (E) and its quantification (F). Note a significant decrease in the surface IGF1Rα level by the KIF1B deficiency. Tot, total lysate; Sur, surface fraction. n = 8. **, P < 0.01, ***, P < 0.001, one-sided paired Welch’s t test. (G and H) Fluorescent micrographs of DIV3 hippocampal neurons of the indicated genotypes transfected with EGFP and cultured with or without 10 nM IGF-I in the medium (G) accompanied with the statistical analysis (H). The axons of Kif1b^+/+ neurons with 10 nM IGF-I in the culture were significantly longer than the ones without IGF-I. However, the IGF-I stimulation was less effective in Kif1b^−/− neurons. Bars, 25 µm. n = 29–40. *, P < 0.05; ns, P > 0.05, one-sided unpaired Welch’s t test.