Fig. 2.

Rassf5 is required for neuronal polarity. (A) Neurons from the hippocampus of E18 rat embryos were fixed at stage 2 and stage 3, and stained with the anti-Rassf5 antibody. A higher magnification of the axon at stage 3 is shown in the lower-left corner. Scale bars: 10 µm. (B) HEK 293T cells were transfected with vectors for GFP, HA–Rassf5A, shRNA-resistant HA–Rassf5A (R5M) and an shRNA against Rassf5 (R5-sh) or pSM2 (control), as indicated. The expression of Rassf5A was analyzed by western blotting (WB) using an anti-HA antibody. Staining for GFP confirmed comparable transfection efficiencies and protein loading. (C) Hippocampal neurons were transfected at 0 DIV with vectors for GFP (green), shRNA-resistant Rassf5A (R5M) and an shRNA against Rassf5 or pSM2 (control). Neurons were fixed at 3 DIV and stained with the Tau-1 antibody (blue in overlay) and an anti-MAP2 antibody (red in overlay). Scale bars: 50 µm. (D) The percentage of unpolarized neurons without an axon (0, white), polarized neurons with a single axon (1, gray) and neurons with multiple axons (>1, black) is shown. Values are the mean±s.e.m. (three transfections, n>150); ns, non-significant; ***P≤0.001 compared with control (GFP+pSM2); two-way ANOVA.