JIP3 must be transported to the axon tips to exert its effect on axon elongation.
A, quantitation of the distribution of JIP3-EGFP and JIP3ΔΔ-EGFP in distal axons of hippocampal neurons at DIV5 (n = 3, ***, p < 0.001, versus the JIP3-EGFP group; Student's t test). B, hippocampal neurons (DIV5) transfected with pCAGGS-EGFP and scramble or KIF5 siRNA were stained with anti-GFP (green), anti-KIF5 (gray), and anti-α-tubulin (blue) antibodies. Arrows point to the GFP-positive neurons. Scale bar, 40 μm. C, hippocampal neurons were transiently transfected with scramble or KIF5 siRNA. At 5 days after transfection, cells were lysed and immunoblotted with anti-KIF5 and anti-α-tubulin antibodies. D, quantitation of the distribution of JIP3-EGFP in distal axons of hippocampal neurons at DIV5 when co-transfected with scramble or KIF5 siRNA (n = 3, ***, p < 0.001, versus the scramble group; Student's t test). E, quantitation of the axon length of the neurons co-transfected with empty vector or JIP3-EGFP construct and scramble or KIF5 siRNA (n = 3, *, p < 0.05, versus the control + scramble group; NS, no significance; one-way analysis of variance).