anti vegf a165  (R&D Systems)

Journal: Cells

Article Title: Retinal Pigment Epithelium-Secreted VEGF-A Induces Alpha-2-Macroglobulin Expression in Endothelial Cells.

doi: 10.3390/cells11192975

Figure Lengend Snippet: Figure 3. VEGF-A-dependent induction of A2M expression in ECs by RPE conditioned medium. (a–c) Positive correlation between hfRPE TER, VEGF-A levels in hfRPE basolateral conditioned media (CM) and its ability to induce A2M expression in ECs. After 1 or 4 weeks in culture (W1 and W4, respectively), hfRPE TER was measured ((a), n = 6, t-test) and basolateral conditioned media were collected and used to induce ECs, which were tested for A2M expression by real-time PCR 24 h after induction ((b), n = 6, ANOVA). Collected media were also used for the assessment of VEGF-A levels by ELISA ((c), n = 6, t-test). (d) Real-time PCR showing very low VEGR2 expression in 293T and HeLa cells compared to ECs (n = 3, ANOVA). (e,f) The VEGFR2 inhibitor SU5614 (e) and a VEGF-A blocking antibody (f) inhibit the induction of A2M expression in ECs by hfRPE conditioned medium (24 h) in a dose-dependent manner ((e), n = 3, ANOVA; *** and **, vs. 0 µM SU5614; (f), n = 5, ANOVA; *** and **, vs. mock). (g) Cell viability of ECs after a 24 h exposure to RPE conditioned medium in the absence (control) or presence of 2 µM SU5614, 1 µg mL−1 control or anti-VEGF-A antibody (control, n = 9; rest, n = 6; ANOVA). (h) Real-time PCR showing A2M mRNA levels in ECs after incubation with recombinant VEGF-A for different times (n = 6, ANOVA). ***, 0 h vs. 8 h, 16 h and 24 h. (i) Luciferase assays showing transcriptional activation of human A2M promoter after incubation of transiently transfected ECs with recombinant VEGF-A for 8 h (n = 6, t-test).

Article Snippet: SU5614 (Sigma-Aldrich), control IgG (cat. AB-108-C, R&D Systems, Minneapolis, MN, USA) or anti-VEGF-A165 antibody (cat. AF-293-NA, R&D Systems) were added to the conditioned media as indicated.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Blocking Assay, Control, Incubation, Recombinant, Luciferase, Activation Assay, Transfection

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A ) Serum-starved Bsp73AS (AS) or Bsp73ASs6 (ASs6) cells were incubated with a CD44v6 antibody (1.1ASML) , a v6 peptide (14mer) or a control peptide prior to induction with biotinylated HGF and subsequent detection of binding using FITC–streptavidin. All samples were analysed in a FACScan flow cytometer. The FITC–streptavidin negative control is shown in blue, HGF-induced samples either non-treated or treated with CD44v6-specific blocking reagents are shown in green. Representative pictures from one experiment are shown. Three independent experiments per condition were performed giving the same results. ( B ) Serum-starved T47D and T47D/Met cells were pretreated either with a CD44v6 antibody (VFF18), or a v6 peptide (14mer) prior to induction with biotinylated HGF. Expression of CD44v6 and CD44s.ectodomains (labelled as Ecd) was achieved by transfection of the expression constructs using Lipofectamine. The cells were then subjected to FACS analysis 48 h after transfection. Binding of biotinylated HGF was detected using FITC labelled streptavidin. All samples were analysed in a FACScan flow cytometer. Cells exclusively incubated with FITC–streptavidin were used as a negative control (purple). Cells treated with HGF alone, or pretreated with the peptides, antibody or transfected with the ectodomains are represented in green. Representative pictures from one experiment are shown. Three independent experiments were performed giving the same results. ( C ) Serum-starved HUVEC and ASs6 cells were pre-incubated either with a v6 peptide (5mer) or a control peptide as indicated and induced with VEGF 165 . Binding of VEGF 165 to the cells was detected by means of a specific antibody against VEGF and a secondary PE-labelled antibody and subsequently analysed in a FACScan flow cytometer. Cells exclusively incubated with the PE-labelled antibody were used as a negative control (purple). Cells treated with VEGF 165 or additionally pre-incubated with the peptides and probed with the antibodies are represented in green. Representative pictures from one experiment are shown. Three independent experiments were performed giving the same results.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Incubation, Control, Binding Assay, Flow Cytometry, Negative Control, Blocking Assay, Expressing, Transfection, Construct

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A ) For the cellular ELISA serum-starved AS, ASs6 and HT29 cells were induced with biotinylated HGF. The binding of the growth factor to the cells was detected by means of an avidin–HRP conjugate and ABTS solution and subsequently analysed in an ELISA reader. Experimental data are reported as mean ± S.D. of three independent experiments (* P < 0.05, ** P < 0.005; Student's t -test). ASs6 cells were in addition treated with a v6 (5mer) and a control peptide as indicated and induced with biotinylated HGF. ( B ) The AS and ASs6 cells were transfected with a VEGFR-2 expression construct using FuGene. 48 h after the transfection the cellular ELISA was performed with AS, ASs6 and HUVEC cells as described above and the cells were induced with biotinylated VEGF 165 .

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Avidin-Biotin Assay, Control, Transfection, Expressing, Construct

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A – C ') The nickel pull-down assays were performed with the histidine-tagged ectodomains of CD44 (CD44s and CD44v6). The CD44 ectodomains were coupled to Ni-NTA beads (labelled as NiB) and incubated with the growth factors HGF, VEGF 165 and TGF-α as indicated. ( A , B , C ) Binding of the ligands to the histidine-tagged CD44 ectodomains was analysed in Western blots by means of specific antibodies against HGF, VEGF and TGF-α. As loading controls, the respective growth factors as well as the CD44 ectodomains were loaded directly on the gel. ( A ', B ', C ') To control the equal loading of the ectodomains the membranes were stripped and reprobed with a specific antibody against histidine for detection of the histidine-tagged CD44 proteins. The Western blots were performed at least three times and gave similar results. A representative blot is shown.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Incubation, Binding Assay, Western Blot, Control

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: ( A – F ) Using MST the K d was determined whenever binding was observed (fitting curves in blue). ( A ) Fluorescently labelled VEGF 165 was titrated against varying concentrations of the CD44v6 ectodomain ( K d =51±9 nM) or ( B ) the CD44s ectodomain. ( C ) Titration of serial dilutions of the CD44v6 ectodomain ( K d =610±90 nM) or ( D ) the CD44s ectodomain ( K d =130±300 nM) to fluorescently labelled HGF. ( E ) Fluorescently labelled CD44v6 ectodomain was titrated against serial dilutions of TGF-α or ( F ) against the Met ectodomain (Met 928 ). Binding affinities were calculated from at least three different experiments.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Binding Assay, Titration

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: (A–D) Dissociation constants were determined via FCS by titrating the respective CD44 isoform against the fluorophore-labelled growth factor. The study on VEGF 165 -ATTO 647N binding to ( A ) CD44v6 and ( B ) CD44s revealed dissociation constants of 65±8 nM and 1300±600 nM, respectively. The same experiment was conducted with HGF-ATTO 647N yielding a ( C ) K d =1830±60 nM for CD44v6 and ( D ) K d =5000±3000 nM for CD44s.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Binding Assay

Journal: Bioscience Reports

Article Title: Direct binding of hepatocyte growth factor and vascular endothelial growth factor to CD44v6

doi: 10.1042/BSR20150093

Figure Lengend Snippet: Overview of binding affinities as determined with MST, FCS and FA The dissociation constants ( K d ) shown are the average values of N experiments carried out for each pair of binding partners.

Article Snippet: The antibodies used in the present study were directed against rat CD44v6 (1.1ASML, described in [ ]) and human CD44v6 (VFF18; Boehringer Ingelheim), Erk-1 (K-23; Santa Cruz), phospho-p44/42 MAPK (Thr202/Tyr204; Cell Signaling Technology), penta-His (Qiagen), human HGF (AF-294-NA; R&D Systems), rat Met (B2; Santa Cruz), human Met (25H2; Cell Signaling Technology) phospho-Met (Tyr1234/1235; Cell Signaling Technology), human TGF-α (transforming growth factor alpha; Peprotech), VEGFR-2 (A3; Santa Cruz) and human VEGF-A165 (VEGF 165 ) (AF-293-NA; R&D Systems).

Techniques: Binding Assay, Protein Binding

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis Via VEGFR-2 Without Significant Enhancement of Vascular Permeability and Inflammation

doi: 10.1161/01.atv.0000233336.53574.a1

Figure Lengend Snippet: Figure 1. Generation of Tg mice. A, Transgene ratio and mortality of different Tg founders. B, ELISA detec- tion of the average protein expression level of VEGF- A165, chimera9, and chimera33 in the dorsal skin of cor- responding Tg mice at the stage of postnatal day 5 (n5 per group). *P0.01, **P0.005 vs VEGF-A165.

Article Snippet: The primary antibodies were VEGF C-1 against human VEGF-A165, PlGF C-20 against chimeric VEGF-ENZ7/PlGF (Santa Cruz Biotechnology), anti-human VEGF-C antibody (R&D System), anti-VEGFENZ7, anti-human VEGFR-1 and VEGFR-2 antibody (generated by our laboratory), and anti-human VEGFR-3 antibody (from Dr. Kari Alitalo).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis Via VEGFR-2 Without Significant Enhancement of Vascular Permeability and Inflammation

doi: 10.1161/01.atv.0000233336.53574.a1

Figure Lengend Snippet: Figure 3. VEGF-ENZ7/PlGF induces angiogenesis with low inflammation. A, Histological analysis of the inflammatory condition around the lower lip of a WT mouse and VEGF-A165 Tg mouse. Specimen from VEGF-A165 Tg mouse showed keratinocyte hyperplasia (red arrowhead), papilloma (black arrowhead), vessel dilation (black arrow), inflammatory cell infiltration (white arrowhead), and tissue edema (red arrow). B, Inflammatory cell infiltration in the ear skin of Tg mice examined by immunofluorescence staining. Left panels show the staining results of CD11b (green) merged with Gr-1 (red), and right panels are the staining of F4/80 (green) merged with Gr-1 (red). Magnification is 400. Scale bar is 50 m. C, Quantification of monocyte/macrophage recruitment. *P0.005, **P0.0002 vs WT. Inflammatory cells with double positive staining signal were calculated from 5 different regions for each sample. D, RT-PCR analysis of the expression level of proangiogenic factors and proinflammatory cytokines. The lane “WT” following each Tg mouse was the corresponding WT littermate.

Article Snippet: The primary antibodies were VEGF C-1 against human VEGF-A165, PlGF C-20 against chimeric VEGF-ENZ7/PlGF (Santa Cruz Biotechnology), anti-human VEGF-C antibody (R&D System), anti-VEGFENZ7, anti-human VEGFR-1 and VEGFR-2 antibody (generated by our laboratory), and anti-human VEGFR-3 antibody (from Dr. Kari Alitalo).

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis Via VEGFR-2 Without Significant Enhancement of Vascular Permeability and Inflammation

doi: 10.1161/01.atv.0000233336.53574.a1

Figure Lengend Snippet: Figure 5. VEGF-ENZ7/PlGF-induced blood vessels are coated with well organized SMC. Whole-mount staining of SMA (green) in the ear skin of WT mouse, VEGF-A165, VEGF-ENZ7, and VEGF-ENZ7/PlGF Tg mice. Left panels are arterioles and right panels are venules. SMCs loosely attached to the arteriole (arrows) and big pore structures on the surface of the venule (arrow heads) were found only in VEGF-A165 Tg mice. Magnifica- tion is 400. Scale bar is 20 m.

Article Snippet: The primary antibodies were VEGF C-1 against human VEGF-A165, PlGF C-20 against chimeric VEGF-ENZ7/PlGF (Santa Cruz Biotechnology), anti-human VEGF-C antibody (R&D System), anti-VEGFENZ7, anti-human VEGFR-1 and VEGFR-2 antibody (generated by our laboratory), and anti-human VEGFR-3 antibody (from Dr. Kari Alitalo).

Techniques: Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis Via VEGFR-2 Without Significant Enhancement of Vascular Permeability and Inflammation

doi: 10.1161/01.atv.0000233336.53574.a1

Figure Lengend Snippet: Figure 6. VEGF-ENZ7/PlGF is the ligand specific to VEGFR-2. Different ligands including human VEGF-A165, VEGF-C, VEGF- ENZ7, chimera9, and chimera33 were used to stimulate the auto- phosphorylation of VEGFR-1, VEGFR-2 (A), and VEGFR-3 (B) at a concentration of 100 ng/mL. (C) Neuropilin-1 binding assay. P-Tyr indicates anti-phosphorylated tyrosine.

Article Snippet: The primary antibodies were VEGF C-1 against human VEGF-A165, PlGF C-20 against chimeric VEGF-ENZ7/PlGF (Santa Cruz Biotechnology), anti-human VEGF-C antibody (R&D System), anti-VEGFENZ7, anti-human VEGFR-1 and VEGFR-2 antibody (generated by our laboratory), and anti-human VEGFR-3 antibody (from Dr. Kari Alitalo).

Techniques: Phospho-proteomics, Concentration Assay, Binding Assay