VEGF165 reduces levels of cell-surface Npn-1 and induces its internalization. (A) (Left) Npn-1 detected on endothelial cells incubated (25°C, 1 hour) with VEGF165 (0-100 ng/mL) or VEGF121 (0 or 100 ng/mL). (Right) VEGFR-2 detected on endothelial cells incubated with VEGF165 (0 or 25 ng/mL) or VEGF121 (0 or 25 ng/mL). Shaded graphs reflect control staining. (B) Npn-1 detected on endothelial cells stimulated (25°C, 1 hour) with VEGF165 (250 ng/mL) plus heparin (0-400 ng/mL). Unstimulated cells: dotted line. (C) Temperature dependency of VEGF165-induced surface Npn-1 reduction. Endothelial cells were incubated with VEGF165 (25 ng/mL) and heparin (2 μg/mL) at 4°C, 25°C, or 37°C for 5, 15, 30, or 60 minutes. Results reflect mean percent signal intensities with stimulation compared with no stimulation. (D) Npn-1 detected on endothelial cells cultured (37°C, 30 hours) with 25 ng/mL VEGF165, VEGF121, or FGF-2. (E) Npn-1 detected on the endothelial-cell–surface (left) and after cell permeabilization (right). Cells were incubated (25°C, 1 hour) with VEGF165 (0, 25, or 250 ng/mL). (F) Npn-1 is internalized after stimulation with VEGF165. Endothelial cells grown on fibronectin-coated glass slides were incubated (37°C; 0, 5, 30, or 60 minutes) with VEGF165 (25 ng/mL). Npn-1 was visualized under an LSM510 confocal microscope equipped with a Plan-Neofluar 40 ×/1.3 objective lens (Carl Zeiss, Thornwood, NY). Images reflect the merging of fluorescent slice and differential interference contrast (DIC) images. Images were imported into Adobe Photoshop 6.0 (Adobe Systems) for processing. Scale bar, 20 μm.
