Figure 7.

Nbs inhibit ephrin-B3-EphA4-mediated growth-cone collapse. Nb 53 and Nb 39 (1 μm) reversed ephrin-B3-mediated growth-cone collapse. A, E17.5 cortical neurons were first treated at 24 h after culture with 1 μm or 30 μm KYL peptide or 1 μm Nb for 30 min and then stimulated with 1 μg/ml preclustered ephrin-B3-Fc or Fc as a control for 30 min in the presence of the KYL peptide or the Nb. A control condition without any kind of treatment was also scored (control). Cells were stained with Alexa Fluor 555-conjugated phalloidin and DAPI. Growth-cone collapse was determined for every neuron by analyzing the tip of only the longest neurite (white arrowheads). Scale bar, 10 μm. B, percentage of growth-cone collapse was determined in every experimental condition by assessing 40–160 neurons under a Zeiss fluorescence microscope. Percentage of collapse is expressed as the mean of collapse of six independent experiments ± S.D. and was compared between different conditions by one-way ANOVA followed by Tukey's multiple comparisons post hoc test. ***, p < 0.001, as compared with control and Fc conditions. #, p < 0.05; ##, p < 0.01 as compared with the ephrin-B3 condition.