HUVEC, Hec50, and KLE cells were grown to 70–80% confluence and serum-starved in Opti-MEM for one hour prior to transfection. Transfection of 1 µg alkaline phosphatase (AP)-tagged transforming growth factor (TGF)-α were performed according to manufacturer’s protocols using Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA). Cells were starved for at least four hours before the stimulation. Recombinant human vascular endothelial growth factor (VEGF-A) was obtained from R&D Systems (Minneapolis, MN, USA) and used at a concentration of 100 ng/mL. The concentration of bevacizumab was 1 µg/mL. PMA (phorbol-12-myristate-13-acetate) and the AP-tagged TGF-α plasmid have been previously published [33 (link)]. Evaluation of AP activity was determined by the colorimetric assay as described previously [34 (link)]. Briefly, the ratio between the total AP activity in the supernatant and the total AP activity in the cell lysate plus the supernatant was computed for normalization. The presented ratios reflect the relative proteolytic activity of a given sheddase toward the AP-tagged ligand TGF-α. No AP activity was detected in the conditioned media of untransfected cells.
Recombinant human vascular endothelial growth factor vegf a
Manufactured by R&D Systems
Sourced in United States
Recombinant human vascular endothelial growth factor (VEGF-A) is a protein that promotes the growth and development of blood vessels. It is a key regulator of angiogenesis, the process of new blood vessel formation.
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2 protocols using recombinant human vascular endothelial growth factor vegf a
1
Quantifying Sheddase Activity on TGF-α
2
Alkaline Phosphatase-Mediated TGF-α Shedding Assay
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HUVEC, Hec50, and KLE cells were grown to 70-80% con uence and serum-starved in Opti-MEM for one hour prior to transfection. Transfection of 1 µg alkaline phosphatase (AP)-tagged transforming growth factor (TGF)-α were performed according to manufacturer's protocols using Lipofectamine2000 (Invitrogen, Carlsbad, CA). Cells were starved for at least four hours before the stimulation. Recombinant human vascular endothelial growth factor (VEGF-A) was obtained from R&D Systems (Minneapolis, MN) and used at a concentration of 100 ng/ml. The concentration of bevacizumab was 1µg/ml. PMA (phorbol-12-myristate-13-acetate) and the AP-tagged TGF-α plasmid have been previously published [21] . Evaluation of AP activity was determined by colorimetric assay as described previously [22] . Brie y, the ratio between the total AP activity in the supernatant and the total AP activity in the cell lysate plus supernatant was computed for normalization. The presented ratios re ect the relative proteolytic activity Loading [MathJax]/jax/output/CommonHTML/fonts/TeX/fontdata.js of a given sheddase toward the AP-tagged ligand TGF-α. No AP activity was detected in the conditioned media of untransfected cells.
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