Figure 5.

The effect of Rnd1 antisense treatment on the morphology of hippocampal neurons. Hippocampal cultures at 15 DIV were treated for 2 d with sense or antisense oligonucleotide and harvested (Aa, Ab) or fixed (Ac, Ad, B) at 17 DIV. Aa, P100 fraction (20 μg of total protein) of harvested cells was immunoblotted with anti-Rnd1 antibody (top) or anti-α-tubulin antibody (bottom). Ab, Quantification of Rnd1 protein level of sense- or antisense-treated neurons. The level of Rnd1 protein was normalized to that of α-tubulin and expressed as fold of the value of sense-treated neurons. Ac, Ad, Immunofluorescence microscopy was shown for EGFP-F-expressing hippocampal neuron treated with sense (c) or antisense (d) oligonucleotide. EGFP-F and Rnd1 labeling were shown in the top and middle panels, respectively. Bottom panels (Merge) show the superposition of the top and middle images. Note that Rnd1 immunostain is faint in antisense-treated neuron but is obvious in sense-treated neuron. B, Immunofluorescence microscopy was shown for EGFP-expressing hippocampal neuron treated with sense (a) or antisense (b) oligonucleotide. EGFP and PSD-95 labeling were shown in the top and middle panels, respectively. Bottom panels (Merge) show the superposition of the top and middle images. Arrows indicate mature-shaped spines, and arrowheads indicate headless protrusions. Another example is shown in a′ for sense-treated neuron or in b′ for antisense-treated neuron. Scale bars, 5 μm.