Figure 8. α_Vβ₃-Fc induces Thy-1/Src co-distribution and Src inhibition in cortical neurons.

Mature cortical neurons were treated with supernatants containing α_Vβ₃-Fc fusion protein (α_Vβ₃-Fc) or with α_Vβ₃-Fc-depleted supernatants (DS) for 15–75 minutes. (A) After 15 minutes, neurons were fixed and immunostained for Thy-1 (green) or total Src kinase (red). Samples were photographed using a conventional fluorescence microscope. Clusters were identified creating a 1-bit mask for each fluorescent channel using Image J software. Black arrows indicate Thy-1 clusters co-distributed with Src clusters. Green arrows indicate Thy-1-only, red arrows Src-only clusters. (B) Quantification of cluster distribution as indicated. **P<0.01, *P<0.05 compared with number of Thy-1/Src co-distributed clusters in DS. #P<0.05 compared to Thy-1/Src co-distributed clusters in α_Vβ₃-Fc. Results averaged from three independent experiments analyzing 5 axonal segments per experiment are shown. (C) At different time points, neurons were lyzed and analyzed by Western blotting. Blots for Src phosphorylated at tyrosine 527 (Src^pTyr527) and total Src (Src^total) are shown. (D) Numerical values were obtained by scanning densitometry from three independent experiments and are expressed as phosphorylated/total Src ratio for each time point. *P<0.05 compared to time = 0. n.s.d. indicates non-significant differences compared to time = 0.