Figure 4
Figure 4. Characterization and functional consequences of G18 binding to NRP1 and SREC-I. (A) Analysis of biotin-G18 binding to NRP1-Fc and NRP2-Fc. Top panel, binding of biotin-G18 to BSA-, IgG1-, NRP1-, or NRP2-coated wells detected by ELISA-based measurement of streptavidin-HRP. Bottom panel, detection of coated proteins by anti-IgG1 antibody detected by ELISA. The results reflect the means ± SD of triplicate wells. (B) Binding of biotin-G18 to NRP1. Biotin-A18, -T18, -G18, or -C18 (0.25, 1, 4, 16 μg/mL) was added to NRP1-coated wells. Bound biotin-olionucleotides were detected by ELISA-based measurement of streptavidin-HRP. The results reflect the means ± SD of 3 experiments. (C) Biotin-G18 binding to NRP1 is selectively blocked by nonbiotin-G18. Biotin-G18 (1 μg/mL) was added to NRP1-coated wells in the presence of G18, A18, T18, or C18 (100 μg/mL). (D) Characterization of NRP1 binding to immobilized G18. Plasmon resonance (Biacore)-generated sensorgrams showing a kinetic analysis of NRP1 binding to G18. Biotin-G18 was immobilized onto the sensor chip. NRP1/Fc (2.5, 5, 7.5, 10, or 20nM) was passed over the sensor surface. Representative results from 4 independent experiments are shown. (E) Binding of biotin-G18 to SREC-I. Biotin-A18, -T18, -G18, or -C18 (0.25, 1, 4, or 16 μg/mL) was added to SREC-I–coated wells. Bound biotin-olionucleotides were detected by ELISA-based measurement of streptavidin-HRP. The results reflect the means ± SD of 3 experiments. (F) G18 bridges NRP1 to SREC-I. His-tagged NRP1/Fc (1 μg/mL) was added to SREC-I/Fc-coated wells in the presence of A18, T18, G18, or C18 (0, 0.25, 1, 4, or 16 μg/mL). Bound His-tagged NRP1/Fc was detected by HRP-conjugated anti-His Ab. The results reflect the means ± SD of 3 experiments. (G) G18 induces the coordinate internalization of NRP1 and SREC-I. HUVECs were incubated with or without sG18 (16 μg/mL at 37°C for 1 hour). After fixation and permeabilization, cells were stained with anti-NRP1 mAb (green), anti-SREC-I Ab (red), and DAPI (blue) and were examined by confocal microscopy. Top panels are from cells incubated in medium alone; bottom panels are from cells incubated with sG18. Scale bar, 20 μm.

Characterization and functional consequences of G18 binding to NRP1 and SREC-I. (A) Analysis of biotin-G18 binding to NRP1-Fc and NRP2-Fc. Top panel, binding of biotin-G18 to BSA-, IgG1-, NRP1-, or NRP2-coated wells detected by ELISA-based measurement of streptavidin-HRP. Bottom panel, detection of coated proteins by anti-IgG1 antibody detected by ELISA. The results reflect the means ± SD of triplicate wells. (B) Binding of biotin-G18 to NRP1. Biotin-A18, -T18, -G18, or -C18 (0.25, 1, 4, 16 μg/mL) was added to NRP1-coated wells. Bound biotin-olionucleotides were detected by ELISA-based measurement of streptavidin-HRP. The results reflect the means ± SD of 3 experiments. (C) Biotin-G18 binding to NRP1 is selectively blocked by nonbiotin-G18. Biotin-G18 (1 μg/mL) was added to NRP1-coated wells in the presence of G18, A18, T18, or C18 (100 μg/mL). (D) Characterization of NRP1 binding to immobilized G18. Plasmon resonance (Biacore)-generated sensorgrams showing a kinetic analysis of NRP1 binding to G18. Biotin-G18 was immobilized onto the sensor chip. NRP1/Fc (2.5, 5, 7.5, 10, or 20nM) was passed over the sensor surface. Representative results from 4 independent experiments are shown. (E) Binding of biotin-G18 to SREC-I. Biotin-A18, -T18, -G18, or -C18 (0.25, 1, 4, or 16 μg/mL) was added to SREC-I–coated wells. Bound biotin-olionucleotides were detected by ELISA-based measurement of streptavidin-HRP. The results reflect the means ± SD of 3 experiments. (F) G18 bridges NRP1 to SREC-I. His-tagged NRP1/Fc (1 μg/mL) was added to SREC-I/Fc-coated wells in the presence of A18, T18, G18, or C18 (0, 0.25, 1, 4, or 16 μg/mL). Bound His-tagged NRP1/Fc was detected by HRP-conjugated anti-His Ab. The results reflect the means ± SD of 3 experiments. (G) G18 induces the coordinate internalization of NRP1 and SREC-I. HUVECs were incubated with or without sG18 (16 μg/mL at 37°C for 1 hour). After fixation and permeabilization, cells were stained with anti-NRP1 mAb (green), anti-SREC-I Ab (red), and DAPI (blue) and were examined by confocal microscopy. Top panels are from cells incubated in medium alone; bottom panels are from cells incubated with sG18. Scale bar, 20 μm.

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