Fig. 4. Reelin promotes dendritic spine formation through Ras signaling in primary hippocampal neurons. (A) Primary hippocampal neurons were transfected with GFP plasmid on DIV 19 and treated with vehicle (Mock medium) or 1.4 μM Reelin on DIV 20 for 24 h. On DIV 21, the neurons were immunostained with an anti-RasGRF1 antibody, and the puncta numbers were measured (vehicle: n=32 dendrites from 6 neurons, Reelin: n=38 dendrites from 7 neurons). (B, C) Primary hippocampal neurons were treated as described above and immunostained with anti-p-ERK and anti-p-CREB antibodies on DIV 21, and the puncta numbers were measured (p-ERK, vehicle: n=40 dendrites from 6 neurons, Reelin: n=46 dendrites from 9 neurons, p-CREB, vehicle: n=32 dendrites from 8 neurons, Reelin: n=38 dendrites from 8 neurons). (D) Primary hippocampal neurons were transfected with GFP plasmid and HA plasmid (control plasmid for RasN17) or GFP plasmid and RasN17 (Ras inactivator) on DIV 19 and treated with vehicle (Mock medium) or 1.4 μM Reelin on DIV 20 for 24 h. Then, dendritic spine density was measured (GFP plasmid+HA plasmid+vehicle: n=18 dendrites from 9 neurons, GFP plasmid+HA plasmid+Reelin: n=28 dendrites from 12 neurons, GFP plasmid+RasN17+Reelin: n=19 dendrites from 7 neurons) *p<0.05, **p<0.01, ***p<0.001. Scale bar=10 μm.
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