Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin enhances VEGF binding to heparin and heparin binds VEGF–Avastin complexes and enhances VEGF binding to Avastin. A, streptavidin-coated 96-well microtiter plates were coated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml) and incubated with 125I-VEGF (10 ng/ml) alone or with Avastin at the concentrations indicated (0–100 nm) for 2 h at 4 °C. 125I-VEGF bound to the plates was extracted and counted, and the average of triplicate determinations ± S.D. is presented. ANOVA was performed, and the conditions that showed a statistically significant increase in binding are indicated by *. B, Avastin was incubated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml)-coated plates in the presence and absence of VEGF (10 ng/ml) for 2 h at 4 °C, and then an ELISA protocol was used to detect bound Avastin. The average values of triplicate determinations ± S.D. are shown. Only conditions where VEGF and Avastin were incubated with biotin–heparin-coated plates produced a statistically significant signal above background. C, to understand whether heparin influences VEGF binding to Avastin, Avastin (0.5 nm) or Fc-FGFR1 (0.5 nm)-coated protein A plates were incubated with 125I-VEGF (10 ng/ml) and the indicated concentration of heparin (0–100 μg/ml) for 2 h at 4 °C. The 125I-VEGF bound to the surface was extracted and measured. The average of triplicate values ± S.D. is presented. All concentrations of heparin tested produced a statistically significant increase in VEGF binding to Avastin.

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Produced, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin's capacity to inhibit VEGF binding to FN is reduced at acidic pH due to decreased Avastin–VEGF binding. A, 125I-VEGF (10 ng/ml) ± Avastin was incubated with FN-coated plates in binding buffer at the indicated pH values (5–8) for 2 h at 4 °C. After the incubation, 125I-VEGF bound to FN was extracted and counted. The average of triplicate values ± S.D. is shown. * indicates a statistically significant difference between VEGF binding in the presence and absence of Avastin; N.S. means not significant. B, values of 125I-VEGF bound in the presence and absence of Avastin were used calculate % VEGF binding inhibition at each pH using the equation: % VEGF binding inhibition = (VEGF bound (absence of Avastin) − (presence of Avastin)/VEGF bound in the absence of Avastin) × 100. C, 125I-VEGF (10 ng/ml) binding to Avastin (0.5 nm) bound to protein A plates was measured at various pH values after a 2-h incubation at 4 °C. 125I-VEGF association to Fc-FGFR1 (0.5 nm)-coated plates was also measured to control for nonspecific interactions. The average 125I-VEGF bound (fmol/well) ± S.D. of triplicate determinations is shown. Statistically significant reduction in VEGF binding compared with the value at pH 8 is noted (*).

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin enhances VEGF binding to heparin and heparin binds VEGF–Avastin complexes and enhances VEGF binding to Avastin. A, streptavidin-coated 96-well microtiter plates were coated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml) and incubated with 125I-VEGF (10 ng/ml) alone or with Avastin at the concentrations indicated (0–100 nm) for 2 h at 4 °C. 125I-VEGF bound to the plates was extracted and counted, and the average of triplicate determinations ± S.D. is presented. ANOVA was performed, and the conditions that showed a statistically significant increase in binding are indicated by *. B, Avastin was incubated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml)-coated plates in the presence and absence of VEGF (10 ng/ml) for 2 h at 4 °C, and then an ELISA protocol was used to detect bound Avastin. The average values of triplicate determinations ± S.D. are shown. Only conditions where VEGF and Avastin were incubated with biotin–heparin-coated plates produced a statistically significant signal above background. C, to understand whether heparin influences VEGF binding to Avastin, Avastin (0.5 nm) or Fc-FGFR1 (0.5 nm)-coated protein A plates were incubated with 125I-VEGF (10 ng/ml) and the indicated concentration of heparin (0–100 μg/ml) for 2 h at 4 °C. The 125I-VEGF bound to the surface was extracted and measured. The average of triplicate values ± S.D. is presented. All concentrations of heparin tested produced a statistically significant increase in VEGF binding to Avastin.

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Produced, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin's capacity to inhibit VEGF binding to FN is reduced at acidic pH due to decreased Avastin–VEGF binding. A, 125I-VEGF (10 ng/ml) ± Avastin was incubated with FN-coated plates in binding buffer at the indicated pH values (5–8) for 2 h at 4 °C. After the incubation, 125I-VEGF bound to FN was extracted and counted. The average of triplicate values ± S.D. is shown. * indicates a statistically significant difference between VEGF binding in the presence and absence of Avastin; N.S. means not significant. B, values of 125I-VEGF bound in the presence and absence of Avastin were used calculate % VEGF binding inhibition at each pH using the equation: % VEGF binding inhibition = (VEGF bound (absence of Avastin) − (presence of Avastin)/VEGF bound in the absence of Avastin) × 100. C, 125I-VEGF (10 ng/ml) binding to Avastin (0.5 nm) bound to protein A plates was measured at various pH values after a 2-h incubation at 4 °C. 125I-VEGF association to Fc-FGFR1 (0.5 nm)-coated plates was also measured to control for nonspecific interactions. The average 125I-VEGF bound (fmol/well) ± S.D. of triplicate determinations is shown. Statistically significant reduction in VEGF binding compared with the value at pH 8 is noted (*).

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Inhibition