Decreased IGF1R/PI3K signaling by KIF1Bβ deficiency. (A and B) Immunocytochemistry against pAkt (Thr308) of primary mouse hippocampal neurons of the indicated genotypes at DIV3 with IGF-I stimulation for 0 and 5 min (A) and its quantification at the cell body (B). Bar, 25 µm. n = 8–50. ***, P < 0.001; ns, P > 0.05, one-sided unpaired Welch’s t test. (C and D) Immunocytochemistry against pAkt of IGF-I–stimulated DIV4 mouse hippocampal neurons electroporated with the indicated TurboRFP-conjugated miRNA vectors (C) and its quantification (D). Open boxes in the middle panels in C indicate the axon tips magnified in the rightmost panels. Bars: 20 µm (low-magnification images); 5 µm (high-magnification images). n = 24–56. **, P < 0.01, ***, P < 0.001, one-sided unpaired Welch’s t test. (E) Fluorescent micrographs of DIV3 Kif1b^+/+ and Kif1b^−/− hippocampal neurons transfected with EGFP as a control or Ras V12 IRES EGFP taken using a Plan Apochromat 20×/0.8 objective. Bars, 20 µm. (F) Quantification of axon lengths of Kif1b^+/+ + control (n = 25), Kif1b^+/+ + Ras V12 (n = 22), Kif1b^−/− + control (n = 22), and Kif1b^−/− + Ras V12 (n = 21). Plots and mean ± SD. Two-way ANOVA post hoc Tukey’s test was used. Before ANOVA tests, the normal distribution was tested using Kolmogorov–Smirnov test throughout the figures. Note that Ras V12 IRES EGFP significantly rescued neurite elongation deficits in Kif1b^−/− hippocampal neurons.