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. 2005 May 23;169(4):681–691. doi: 10.1083/jcb.200409115

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Copyright © 2005, The Rockefeller University Press

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Figure 3. — Localization of cleavage sites. (A) Structure of mVEGF₁₆₄ and localization of cleavage sites. Cleavage sites were determined by Edman sequencing, MALDI-TOF MS, μLC/MSⁿ, and μLC/MS/MS of 16 kD and 6 kD. 463 denotes antibody specific to the amino terminus (N-ter) of VEGF; 375 to the carboxy terminus (C-ter) of VEGF. (B) Glycosylated mVEGF₁₆₄ was incubated with MMP3 or with plasmin at the following molar ratios: 8:1, 4:1, 2:1, and 1:1 (VEGF/proteinase). VEGF cleavage was examined by immunoblotting with antibodies 463 and 375. V, VEGF; 22 kD, glycosylated mVEGF₁₆₄ monomer. (C, top) Schematic structure of mVEGF₁₆₄, mVEGF₁₁₃, and mVEGF_Δ108–118 are shown. (bottom, left) Conditioned media from HEK 293T stable clones were examined by immunoblotting with antibody 463. (bottom, right) VEGFR2 phosphorylation was induced in PAE-VEGFR2 cells by 100 ng/ml VEGF. Antibodies against phosphotyrosine (PTyr) and VEGFR2 were used to examine phosphorylation of VEGFR2. (D) VEGFR2 phosphorylation was induced by different concentrations of VEGF as indicated. (E) Mesh-CAM assays were used for the evaluation of VEGF activity on E10 chicken embryos. The first panel illustrates the assay: polymerized, vitrogen-containing growth factor is placed onto CAM, and the angiogenic response (neovessels) is assessed independently of the preexisting CAM vessels. Each pellet contained 1 μg/mesh VEGF. Evaluation was determined 24 h after the application of pellets to CAM surface. Numbers below are the vessel mean/mm² (±SD) obtained from the evaluation of four independent experiments. Bar, 100 μm. (F) Qualitative representation of angiogenesis in mice in response to a Matrigel plug containing different VEGF forms. (E and F) VEGF₁₁₃ showed large, dilated vessels (brackets). In contrast, VEGF_Δ108–118 showed significant sprouting of thin vessels (arrows). Indicated below is the average vascular density/mm² (±SD) from four independent experiments. Vessel diameters shown in brackets are as follows: mVEGF₁₆₄ = 15 μm; mVEGF₁₁₃ = 109 μm (large) and 16 μm (small). Bar, 100 μm.