KIF1Bβ-Y1087C reduced the capacity to complement axonal outgrowth. (A) Fluorescent images of TagRFP-labeled Kif1b^−/− mouse–dissociated hippocampal neurons at DIV3 expressing the indicated constructs for 48 h. Neurons were plated at a cell density of 8.6 × 10⁴ cells/cm² for this assay. Bars, 10 µm. (B) Anti-GFP IB of the lysates of Neuro2A cells expressing the indicated constructs. (C) Quantification of the axon lengths. Mean ± SEM. n = 23–132. ***, P < 0.001, one-way ANOVA with post Dunnett’s multiple comparisons. (D and E) Immunocytochemistry against pAkt (Thr308) of DIV3 hippocampal neurons electroporated with the control miRNA, KIF1A miRNA, and KIF1Bβ miRNA vectors together with or without miRNA-resistant KIF1Bβ WT and Y1087C mutation at DIV0 (D) accompanied with the statistics of the mean pAkt level along the axon per unit length (E). Note that miRNA-immune KIF1Bβ significantly rescued neurite elongation deficits in Kif1b^−/− hippocampal neurons. Arrows indicate axons. Bars, 50 µm. Plots and mean ± SD. Two-sided one-way ANOVA post hoc Tukey’s test was used. Control RNAi, n = 25; KIF1A RNAi, n = 13; KIF1Bβ RNAi, n = 24; KIF1Bβ RNAi + KIF1Bβ WT, n = 14; KIF1Bβ RNAi + KIF1Bβ Y1087C, n = 20. The P values are indicated in the graph.