Cell specificity of NRP1 reduction by DS500. (A) Cell-surface NRP1 in HUVECs, RS4, HS-5, and COS7-NRP1 cells analyzed by flow cytometry. (B) Reduction of cell-surface NRP1 is detected on HUVECs, but not RS4, HS-5, and COS7-NRP1 cells after stimulation with DS500 (0, 0.5, 2, 8 μg/mL, 37°C, 1 hour). Results reflect the relative mean fluorescence intensities with and without stimulation. (C) Uptake of DiO-Ac-LDL (0, 0.25, 1, 4 μg/mL, 37°C, 1 hour) by HUVECs, but not RS4, HS-5, and COS7-NRP1 cells detected by flow cytometry. Open circles indicate cell uptake of DiO-Ac-LDL (4 μg/mL) in the presence of competitor Ac-LDL (100 μg/mL). Results reflect mean fluorescence intensities after background subtraction.