The following commercial antibodies were used: MyHC (MF20, Developmental Studies Hybridoma Bank, University of Iowa), α-tubulin (Sigma-Aldrich), DAPI mounting medium (VECTOR Laboratories, H-1200) and Ang-1 (AF923, R&D Systems). Recombinant mouse Tie-2 Fc Chimera (762-T2-100; R&D Systems), recombinant human Ang-1 (500 ng/mL; R&D Systems), recombinant human Ang-2 (500 ng/mL; R&D Systems), recombinant human VEGF (50 ng/mL; R&D Systems), human basic fibroblast growth factor (5 ng/mL; R&D Systems), and recombinant basic hepatocyte growth factor (500 ng/mL; R&D Systems) were used in cell culture experiments.
Recombinant human vegf
Manufactured by R&D Systems
Sourced in United States, Mongolia
Recombinant human VEGF is a growth factor that promotes the formation of new blood vessels. It is produced using recombinant DNA technology.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Recombinant human bfgf, by R&D Systems (2 mentions)
Recombinant human pdgf bb, by R&D Systems (2 mentions)
P stat3, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
Sunitinib, by Selleck Chemicals (2 mentions)
P vegfr2, by Cell Signaling Technology (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Toluidine blue o, by Merck Group (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Pdgfrβ, by Cell Signaling Technology (2 mentions)
Vegfr2, by Cell Signaling Technology (2 mentions)
Bevacizumab, by Genentech (2 mentions)
Trypsin edta, by Lonza (2 mentions)
Trypsin neutralization solution, by Lonza (2 mentions)
Integrin α1 antibody mab1973, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
44 protocols using recombinant human vegf
1
Vascular Endothelial Cell Culture and Stimulation
2
PBMC Stimulation and FACS Analysis
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After the isolation of PBMCs from a healthy subject, PBMCs were stimulated with recombinant human M-CSF (R&D, Minneapolis, MN) 1 ng/mL or recombinant human VEGF (R&D, Minneapolis, MN) 2 ng/mL for 72 hours. The stimulated PBMCs were stained with the various antibodies described above and analyzed by FACS.
3
Fibroblast Signaling Pathway Modulation
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IPF fibroblasts and non-fibrotic control cells were grown to 80% confluence and were then serum-starved for 24 hours. Cells were pre-incubated for 30 minutes with nintedanib (400 nM) before different stimuli (recombinant human PDGF-BB [10 ng/ml], recombinant human bFGF [10 ng/ml], recombinant human VEGF [10 ng/ml]: R&D Systems; Abingdon, United Kingdom) were added for another 30 minutes. Western blotting was performed as previously described [11 (link)]. Primary antibodies used: PDGFRα, VEGFR1, FGFR1, c-Abelson (c-Abl), extracellular signal-regulated kinase (ERK) 1/2, phosphorylated (pho) PDGFRα/β, pho-VEGFR2, pho-c-Abl, pho-ERK1/2 (Cell Signaling Technology, BioConcept; Allschwil, Switzerland) and pho-FGFR1α (Abcam; Cambridge, United Kingdom).
4
Cell Proliferation Response to Growth Factors
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Cells were seeded (104 cells/ml) in 24-well plates and allowed to attach overnight before being serum starved (0.1% FCS, 24 hours). Cells were then exposed to different stimuli (recombinant human PDGF-BB [R&D Systems]; recombinant human bFGF [R&D Systems]; recombinant human VEGF [R&D Systems]) in the presence and absence of nintedanib (0.001, 0.01, 0.1, 1 μM) for 48 hours before being manually counted (Neubauer hematocytometer).
5
Evaluation of Gefitinib and Sunitinib on HUVEC and Lung Cancer Cells
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Human umbilical vein endothelial cells (HUVECs) were purchased from Sciencell (CA, USA), and human lung cancer cell lines, HCC827 and H3255, were purchased from American Type Culture Collection (VA, USA). HUVECs were cultured in endothelial cell medium (ECM) kit (Sciencell) containing 5% fetal bovine serum (FBS) and 1% endothelial cell growth supplement. The human lung cancer cell lines were cultured in medium 1640 (Thermo Fisher Scientific, MA, USA) containing 10% FBS (Thermo Fisher Scientific), and 100 U/mL penicillin and 0.1 mg/mL streptomycin were added to the medium. All cells were cultured in an incubator at 37°C with 5% CO2.
Gefitinib and sunitinib were purchased from Selleck (TX, USA). CM082 was provided by Betta Pharmaceutica Co., Ltd. (Hangzhou, China). Recombinant human VEGF was purchased from R&D Systems (MN, USA). Antibodies for VEGFR2, p‐VEGFR2, ERK, p‐ERK, AKT, p‐AKT, p‐STAT3, and STAT3 were purchased from Cell Signaling Technology (MA, USA). Antibodies for VEGF‐A, CD31, Ki‐67, β‐actin, and β‐tubulin, and horseradish peroxidase‐conjugated antirabbit/mouse IgG antibodies, were purchased from ABclonal Technology (Wuhan, China).
Gefitinib and sunitinib were purchased from Selleck (TX, USA). CM082 was provided by Betta Pharmaceutica Co., Ltd. (Hangzhou, China). Recombinant human VEGF was purchased from R&D Systems (MN, USA). Antibodies for VEGFR2, p‐VEGFR2, ERK, p‐ERK, AKT, p‐AKT, p‐STAT3, and STAT3 were purchased from Cell Signaling Technology (MA, USA). Antibodies for VEGF‐A, CD31, Ki‐67, β‐actin, and β‐tubulin, and horseradish peroxidase‐conjugated antirabbit/mouse IgG antibodies, were purchased from ABclonal Technology (Wuhan, China).
6
Investigating VEGF-Mediated Angiogenic Signaling
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Fetal bovine serum was purchased from Gibco (Grand Island, NY, USA). Recombinant human VEGF was purchased from R&D Systems (Minneapolis, MN, USA), and MTT was obtained from Sigma (St. Louis, MO, USA). Matrigel was purchased from BD Bioscience (San Jose, CA, USA). LDH assay kits were purchased from Promega (Madison, WI, USA). Drabkin’s reagent kit 525 was obtained from Sigma (St. Louis, MO, USA). Endothelial cell growth supplement (ECGS) was obtained from Upstate Biotechnology (Lake Placid, NY, USA). Anti-phospho-VEGFR2 rabbit pAb and Anti-VEGFR2 rabbit pAb was purchased from Cell Signaling (Danvers, MA, USA). Furthermore, p-ERK1/2 (Tyr 204) rabbit pAb, ERK1/2 mouse mAb, p-PI3 kinase p85α(Tyr 508) rabbit pAb, PI3 kinase p85α mouse mAb, p-AKT (Ser 473) mouse mAb, Akt mouse mAb, p-mTOR (Ser 2448) mouse mAb, mTOR mouse mAb,α-tubulin mouse mAb, and peroxidase-conjugated anti-mouse and anti-rabbit antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
7
VEGFR-2 Signaling Pathway Regulation
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Anti Antiphospho-ERK1/2 (Tyr 204, sc-101761), ERK1/2 (sc-514302), Antiphospho-PI3K-p85α(Tyr 508, sc–12929), PI3K-p85α (sc–1637), p-FAK (Tyr 397, sc-11765-R), FAK (sc-1688), VEGFR-2 (sc-6251) and secondary antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti Antiphospho-VEGFR 2 (Y1175) was purchased from Cell Signaling (Danvers, MA). Beta actin (NB600–501) was purchased from Novvsbio. Iintegrin α2 (ab24697), α1 (ab78479) and β1 (ab30483) were purchased from ABCAM. MTT and toluidine blue O were purchased from Sigma (St Louis, MO). M199, fetal bovine serum (FBS) and other cultured reagents were purchased from Gibco (Grand Island, NY). Endothelial cell growth supplement was purchased from Upstate Biotechnology. Recombinant human VEGF was purchased from R&D Systems (Minneapolis, MN). Aggretin α–chain C-terminus (AACT) 106–136 (CGALEKLTGFRKWVNYYCEQMHAFVCKLLPY) were synthesized by MDBio, Inc., Taiwan. All protocol were approved by Institutional Review of Board, National Taiwan University Hospital or Laboratory Animal Center, College of medicine, National Taiwan University. All experiments were performed in accordance with College of medicine, National Taiwan University regulations and the the written informed consent was obtained from all subjects for human platelet suspension preparation.
8
Transfection and Serum Starvation of CECs
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CECs were transfected with an equal concentration of either control small interfering RNA (siRNA) or EPOR siRNA oligos using Lipofectamine 3000 following the protocol recommended by the manufacturer (Life Technologies, Grand Island, NY, USA). Forty-eight hours after transfection, CECs were serum-starved in Endothelial Basal Media-2 (EBM-2, Lonza, Walkersville, MD, USA) for three hours and then treated with either recombinant human VEGF (25 ng/mL, R&D Systems, Minneapolis, MN, USA), recombinant human EPO (5 U/mL, R&D Systems, Minneapolis, MN, USA) or PBS as vehicle control for 30 min. All treatments were volume-controlled.
9
Dopamine Receptor Modulation of Primary Endothelial Cells
10
Antibody Validation for Cellular Signaling
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Rabbit monoclonal antibody (mAb) against YAP (Cat No. 52771), rabbit mAb against Ki67 (Cat No. 15580), mouse mAb against human CD31 (Cat No. 9498), rabbit mAb against ERG (Cat No. 92513) and rat mAb against BrdU (Cat No. 6326) were purchased from Abcam (Cambridge, United Kingdom). Rabbit mAb against YAP (Cat No. 14074), Rabbit mAb against pYAP (Cat No. 4911) and rabbit mAb against MST1 (Cat No. 14946) were from Cell Signaling Technology (Boston, MA, United States). Mouse mAb against β-actin, Mouse mAb against GAPDH and Mouse mAb against β-tubulin were from Utibody (Tianjin, China). LB100 (Cat No. s7537) was from Selleck (Houston, TX, United States). LatB (Cat No. L5288) was from Sigma Aldrich (St. Louis, MO, United States). Recombinant Human VEGF (Cat No. 293-VE) was from R&D systems (Minneapolis, MN, United States).
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