Figure 1

NMDAR activation rapidly increases Wnt5a in cortical cultures. A. Cellular localization of Wnt5a in neurons. Shown are confocal images of primary cortical neurons after double-fluorescent immunostaining with anti-Wnt5a (red) and anti-synapsin I (green) antibodies. The nucleus was stained by DAPI (blue). B. MSG or NMDA stimulation increased Wnt5a protein. Primary cortical neurons (10 DIV) were treated with 10 μΜ MSG or 50 μΜ NMDA for 15 min. Wnt5a protein was detected by Western blotting. Data in the summary graphs (mean ± SEM) were from three independent experiments (*, p < 0.05; **, p < 0.01; One-way ANOVA). C. NMDA receptor-regulated Wnt5a increase. Primary cortical neurons (10 DIV) were pre-treated with vehicle (Control) or 100 μΜ DAP5 for 30 min, then incubated with 50 μΜ NMDA for 15 min. Wnt5a and p-P70S6K(included as a marker for translation activation) were detected by Western blotting summarized in the graph (n = 3; *, p < 0.05; **, p < 0.01; One-way ANOVA). D. Dynamic expression of Wnt5a protein after NMDA stimulation. Primary cortical neurons (10 DIV) were treated with 50 μΜ NMDA for 0, 5, 15, 30 and 60 min followed by Western blotting analysis of Wnt5a (n = 3; *, P < 0.05; **, p < 0.01; One-way ANOVA). E. NMDA-induced Wnt5a protein secretion. Primary cortical neurons (10 DIV) were treated with 50 μΜ NMDA for 0, 2, 4, 8, 16 and 32 min, and Wnt5a protein in the media was concentrated and detected on immunoblots.