Analysis of NRP1 and SREC-I colocalization and association in the presence of sulfated polysaccharides. (A) Colocalization of SREC-I and NRP1 in the cytoplasm after fucoidan or DS500 stimulation. HUVECs were incubated (37°C, 1 hour) with medium alone, heparin, ChoSul A, fucoidan, or DS500 (8 μg/mL). After fixation and permeabilization, cells were stained for SREC-I (red), NRP1 (green), and DAPI (blue), and examined by confocal microscopy. Scale bar represents 20 μm. Images were acquired and processed as described for Figure 2A. (B) DS500 specifically and dose-dependently promotes binding of NRP1 to SREC-I. NRP1/Fc or control Fc protein (B7-1/Fc) was added to control IgG1-coated wells (●) or SREC-I/Fc–coated wells (○) with or without DS500. Bound NRP1 or control/Fc was measured by ELISA. The results reflect the means (± SD) of 3 experiments. (C) Effect of polysaccharides on the binding of NRP1 to SREC-I. NRP1/Fc (2μg/mL) was added to SREC-I/Fc–coated wells in the presence of the indicated polysaccharide (500 ng/mL). Bound NRP1/Fc was measured by ELISA. The results reflect the means (± SD) of 3 experiments. (D) Transduction of 293 cells with SREC-I confers DiO-Ac-LDL uptake capability and reduces cell-surface levels of NRP1, but not levels of gp130 or CXCR4. 293 cells were transfected with cDNA for SREC-I (red line) or control (blue line). Uptake of DiO-Ac-LDL and cell-surface levels of endogenous gp130, CXCR4, or NRP1 were detected by flow cytometry. Shaded graphs reflect control staining.