Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) The extracellular domain of mouse VEGFR-3 was immobilized on microtiter wells and incubated with the X6 phage display library. Bar graph shows enrichment in the number of phage recovered [in transducing units (TU)] after consecutive rounds of selection (I, II, and III). (*) Round I was not quantified to prevent the loss of phage displaying unique peptides. ( B ) Peptide identified by sequencing phage bound to VEGFR-3 (round III) ( n , number of phages sequenced). ( C and D ) Binding of control phage Fd (white bars) and phage PCAIWF (B, black bars) and WVCSGG (C, black bars) to VEGF receptors and co-receptors immobilized on microtiter wells. ( E and F ) Inhibition of phage PCAIWF (E) or WVCSGG (F) binding to immobilized VEGFR-3 by synthetic peptide PCAIWF or control peptide (CARAC). The minus sign indicates that no synthetic peptide was added to the assay. ( G ) Dose-response assay. Phage PCAIWF was incubated with immobilized VEGFR-3 in the presence of synthetic peptides PCAIWF, PSAIWF, or CARAC (control). Percentage relative to phage binding in the absence of competing peptide. In all cases, bars represent means ± SEM from triplicate plating. Statistics, Student’s t test (** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Incubation, Selection, Sequencing, Binding Assay, Control, Inhibition

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence or absence of VEGF-A or VEGF-C (10 ng/ml). ( B ) Binding of phage PCAIWF to immobilized VEGFR-3 in the presence of increasing concentrations of VEGF-C. Percentage relative to phage binding in the absence of VEGF-C. ( C ) Cartoon showing the three-dimensional structure of the complex VEGF-C (red) bound to VEGFR-2 IgD2-3 (shown in orange and green, respectively) (Protein Data Bank #2X1W). ( D ) Analysis by SDS–polyacrylamide gel electrophoresis of purified recombinant IgD2 and IgD2-3 proteins containing the ligand-binding domain of VEGFR-3. ( E ) Binding of phage PCAIWF to VEGFR-3 and its recombinant Ig domains immobilized on microtiter wells in the presence or absence of the synthetic peptide PCAIWF or its scramble version, IFCAPW (100 μg/ml). Phage binding was quantified by FLISA using an anti-bacteriophage sera. ( F ) Binding of VEGF-C to microtiter wells coated with immobilized recombinant ligand binding domains IgD2 and IgD2-3 of VEGFR-3 in the presence or absence of synthetic peptides PCAIWF and WVCSGG or the scramble control peptide (IFCAPW). For phage experiments (A and B), bars represent mean ± SEM from triplicate plating; for FLISA assays ( E to G ), bars represent means ± SEM from duplicate wells. Statistics, Student’s t test [not significant (N.S.), P > 0.05; * P ≤ 0.05 and *** P ≤ 0.001].

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Binding Assay, Polyacrylamide Gel Electrophoresis, Purification, Recombinant, Ligand Binding Assay, Fluorophore-linked Immunoabsorbent Assay, Control

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Representation of the VEGF family, their receptors, and pattern of interaction. ( B to F ) Recombinant proteins for the human VEGFR-3 (B), VEGFR-2 (C and E), and VEGFR-1 (D and F) extracellular domains were immobilized on microtiter wells and incubated with the human ligands VEGF-C (B and C), PlGF (D), and VEGF-A (E and F) in the presence or absence of synthetic peptides PCAIWF and PSAIWF or the scramble control peptide (IFCAPW). Growth factors bound to the wells were quantified by FLISA using immunospecific antibodies and fluorescent detection. Bars represent means ± SEM from duplicate wells. Statistics, analysis of variance (ANOVA) (Tukey’s multiple comparison test) (* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Recombinant, Incubation, Control, Fluorophore-linked Immunoabsorbent Assay, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Immunoblot analysis of phosphorylated and total forms of ERK1/2 in LECs incubated with VEGF-A, VEGF-C, or FGF (100 ng/ml) in the presence or absence of peptide PCAIWF or scramble (IFCAPW) (30 μg/ml). ( B ) Ratio of fluorescent intensity for phosphorylated and total ERK1/2. Bars represent means ± SEM from three independent measurements of the immunoblot membrane. Two independent experiments were performed with similar results. Bars represent means ± SEM from triplicate readings. Statistics, ANOVA (Tukey’s multiple comparison test) (* P ≤ 0.05).

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: Western Blot, Incubation, Membrane, Comparison

Journal: Science Advances

Article Title: Discovery of pan-VEGF inhibitory peptides directed to the extracellular ligand-binding domains of the VEGF receptors

doi: 10.1126/sciadv.1600611

Figure Lengend Snippet: ( A ) Tube formation by HUVECs in Matrigel induced by VEGF or VEGF-C in the presence or absence of peptide PCAIWF or scramble (500 μg/ml, embedded in the Matrigel layer). ( B ) Number of tubes formed between endothelial cells. Bars represent means ± SEM from triplicate wells. Statistics, Student’s t test (* P ≤ 0.05). Two independent experiments were performed with similar results.

Article Snippet: Antibodies and other reagents were obtained commercially: anti-human VEGF (AF-293-NA), anti-human VEGF-C (AF752), anti-human PlGF (AF-264-PB), anti-human PDGF-BB (AF-220-NA), anti-human FGF-basic (AF-233-NA), anti-mouse/rat NRP-1 (AF566), and anti-mouse/rat NRP-2 (AF567) were from R&D Systems; anti-fd Bacteriophage-Biotin Conjugate (B2661) was from Sigma-Aldrich; secondary antibodies IRDye 680LT Donkey anti-goat IgG and IRDye 680LT Streptavidin were from LI-COR.

Techniques: