DS500 promotes NRP1 internalization and colocalization with Lamp1. (A) DS500 induces NRP1 internalization. HUVECs grown on fibronectin-coated glass slides were incubated with DS500 (8 μg/mL, 37°C, 0-60 minutes). After fixation and permeabilization, cells were stained with anti-NRP1 mAb and examined by an LSM510 confocal microscope equipped with a Plan-Neofluar 40×1/1.3 objective lens (Carl Zeiss). Images reflect the merging of fluorescent slice images of NRP1 (green), DAPI (blue), and differential interference contrast image. Images were imported into Adobe Photoshop 6.0 (Adobe Systems) for processing. Scale bar represents 20 μm. (B) NRP1 colocalizes with Lamp1. HUVECs were incubated with DS500 (8 μg/mL, 37°C, 1 hour). After fixation and permeabilization, cells were stained for NRP1 (green), Lamp1 (red) and DAPI (blue), and examined by confocal microscopy. (C) DS500 reduces protein levels of NRP1. Cell lysates of HUVECs treated with DS500 (2 μg/mL, 37°C, 0-90 minutes) were blotted with anti-NRP1 Ab (top panel) and reblotted with antiactin Ab (bottom panel). Relative ratios of NRP1/actin band intensities are shown in the lower bar graph.