Figure 5

Overexpression of KIF proteins in vivo. (A) Schematic representation of the expression constructs used in this study, and an illustration of the injection site of the ssAAV9 vector. (B) Western blot of HeLa cell lysates co-transfected with constructs expressing tTA under control of the L7 promoter and GFP–2A–KIF3A or GFP–2A–KIF3C under control of the TRE promoter. The expression of KIF3A or KIF3C, together with GFP, was confirmed by the indicated antibodies. Full-length blots are presented in Supplementary Fig. S5. (C) Representative images of Elavl3^−/− Purkinje cells infected with ssAAV9 vectors expressing tTA and GFP, GFP–2A–KIF3A or GFP–2A–KIF3C. Sections were immunostained with anti-GFP antibody. Asterisks and arrows indicate somata of Purkinje cells and axonal spheroids in the granule cell layer, respectively. Scale bar, 10 μm. (D and E) The areas of each spheroid were measured and plotted in the column graph (left) and the line graph (right). (D) The size of spheroids was significantly enlarged by the expression of GFP–2A–KIF3A or GFP–2A–KIF3C compared to controls (GFP) (D left; n = 151, 182, and 95 spheroids from 3, 2, and 2 independent mice, respectively). The frequency of the spheroids with sizes of 21 to 25 μm² was increased compared to controls (D right). (E) The size of spheroids of uninfected Purkinje cells was not changed between groups (n = 292, 153, and 215 spheroids from 3, 2, and 2 independent mice, respectively). *P < 0.05, **P < 0.01 (one-way ANOVA followed by the Bonferroni post hoc test).