Figure 4

Vegf165 activates FLK1 on GFP + endothelial cells in a NRP1-dependent manner. FLK1-eGFP E11.5 + 2 lung explants were exposed to either 3% (A) or 50% (B-D) oxygen in the presence of either anti-NRP1 antibody (D, 10 microgram/ml) or isotype control (B) for 30 minutes, followed by Vegf165 (C,D, 100 ng/ml) for 15 min. Explants were immunostained for phosphorylated FLK1 (pFLK1). Arrows indicate pFLK1 positive (red), GFP positive (green) endothelial cells. E) Quantitation of pFLK1 positive cells/total cells x 100 and F) Quantitation of pFLK1 positive, GFP positive cells/total GFP positive cells x 100 respectively. n = 4 separate experiments with 2–3 lungs/condition/experiment. *p <0.05 vs. 50% + Isotype and 50% + Anti-NRP1 + Vegf165. **p <0.05 vs. 50% + Isotype and 50% + Anti-NRP1 + Vegf165. Photographs at 40x magnification. Bar = 20 microns. White asterisks show airway lumen.