Fig. 7. NRP1 expression and arterial induction by VEGF¹⁶⁴ in vitro. (A-L) PECAM1⁺, ephrin B2-ß-gal^- embryonic endothelial cells isolated by flow cytometry from Efnb2^lacZ/+ knock-in embryos were cultured in 100 pg/ml of VEGF¹²⁰ or VEGF¹⁶⁴ plus 10 ng/ml bFGF for 2 days, and double-labeled with antibodies to either ß-gal (A-F, red) or NRP1 (G-L, red) together with PECAM1 (A-C,G-I, green). Scale bars: 100 µm. (M) Preferential induction of ephrin B2 by VEGF¹⁶⁴. Cells as in A-L were cultured in the indicated concentrations of VEGF¹²⁰ or VEGF¹⁶⁴ for 2 days, followed by double-labeling with anti-PECAM and X-gal. Both the percentage of ECs expressing ephrin B2-lacZ (left) and the total number of ECs (right) were determined. The concentrations of VEGF¹²⁰ were 1.4-fold higher, on a molar basis, then the concentration of VEGF¹⁶⁴ used at each serial dilution. Bars represent mean±s.e.m. Asterisks indicate statistically significant differences (P<0.05) between VEGF¹⁶⁴ and VEGF¹²⁰ (Student's t-test).