Vegfr3

Manufactured by Abcam

Sourced in United Kingdom, United States

VEGFR3 is a receptor tyrosine kinase that binds vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor D (VEGF-D), promoting lymphangiogenesis. It plays a key role in the development and maintenance of the lymphatic vascular system.

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Lab products found in correlation

Lyve 1, by Abcam (4 mentions) Vegf c, by Abcam (2 mentions) Podoplanin, by Abcam (2 mentions) Prox1, by Abcam (2 mentions) P enos, by Cell Signaling Technology (2 mentions) Vegfr 3, by Merck Group (2 mentions) P akt, by Cell Signaling Technology (2 mentions) Metamorph, by Molecular Devices (2 mentions) Vegfr2, by Cell Signaling Technology (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions) β catenin, by Abcam (1 mentions) P akt1, by Abcam (1 mentions) Ephrina1, by Santa Cruz Biotechnology (1 mentions) Epha3, by Santa Cruz Biotechnology (1 mentions) Wnt5a, by Abcam (1 mentions) Lyve 1, by Novus Biologicals (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions) Anti vegfc, by Abcam (1 mentions) Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)

11 protocols using vegfr3

Comprehensive Antibody Panel for Signaling

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The antibodies used in this study were as follows: EGFR (1:50; # ab52894), HER3 (1:25; # ab5470), HER4 (1:150; # ab19391), VEGFR3 (1:100; # ab27278), VEGF-C (1:50; # ab135506), Wnt5a (1:100; # ab72583), Beta-Catenin (1:100; # ab32572), p-Akt1 (1:100; # ab32505), Akt1 (1:50; #ab59380) were purchased from Abcam company, UK. HER2 (1:100, #4290) was purchased from Cell Signaling Technology, Inc., USA. EphrinA1 (1:100; # sc-911) and EphA3 (1:100; # sc-920) were purchased from Santa Cruz Biotechnology, USA. Horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako EnVision, USA).

Padthaisong S., Thanee M., Namwat N., Phetcharaburanin J., Klanrit P., Khuntikeo N., Titapun A, & Loilome W. (2020). A panel of protein kinase high expression is associated with postoperative recurrence in cholangiocarcinoma. BMC Cancer, 20, 154.

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Immunostaining of Lymphatic Endothelial Cells

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LECs were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 at room temperature. After blocking with donkey serum in PBS containing 0.2% Triton X-100 for 0.5 h, the LECs were incubated with primary antibodies against VEGFR3 (Abcam) and LYVE1 (Novus Biologicals) for 1 h at room temperature. After washing three times with PBS, the LECs were incubated for 1 h with a secondary antibody at room temperature. The slides were washed three times with PBS, mounted with Fuoroshield mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI: Sigma), and sealed with nail polish. Slides were observed by confocal microscopy (Carl Zeiss Microscope).

Wang C., Yue Y., Huang S., Wang K., Yang X., Chen J., Huang J, & Wu Z. (2022). M2b macrophages stimulate lymphangiogenesis to reduce myocardial fibrosis after myocardial ischaemia/reperfusion injury. Pharmaceutical Biology, 60(1), 384-393.

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Protein Expression Analysis by Western Blot

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Protein analysis was performed through western blot analysis. The samples were suspended in RIPA lysis buffer (Thermo Fisher, Massachusetts, USA), then kept on ice for 10 min and centrifuged at 16,000 g at 4°C for 10 min. Proteins were collected from supernatants and dissolved with 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and electrophoretically transferred to 0.45 μm PVDF membranes. Transblotting condition was conducted on an ice-cold system at 110 V. The blotted membrane was blocked with PBS containing 0.1% Tween 20 (PBST) and 5% Bovine serum albumin (BSA). The primary antibodies (1:1000, anti-VEGF-C, VEGFR-3, LYVE-1, podoplanin, PROX-1, eNOS, and iNOS; Abcam, Massachusetts, USA) were added to the buffer and incubated at 4°C overnight with gentle shaking. After removing the primary antibodies, the membranes were washed with PBST three times and incubated with horseradish peroxidase (HRP) conjugated secondary antibody (Abcam, Massachusetts, USA) at room temperature. After 2 h, the membranes were washed with PBST three times and the reactive protein bands were visualized by enhanced chemiluminescence detection reagents with exposure to chemiluminescence light film. The protein expression level was quantified by ImageJ software (National Institutes of Health, U.S.A.).

Hsiao H.Y., Mackert G.A., Chang Y.C., Liu J.W., Chang F.C, & Huang J.J. (2023). In vivo vascularized scaffold with different shear-exposed models for lymphatic tissue regeneration. Journal of Tissue Engineering, 14, 20417314231196212.

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VEGFC and VEGFR3 Protein Detection

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Protein was extracted from the hearts using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Duesseldorf, Germany) according to the manufacturer’s instructions. Protein extracts (40 µg) were separated by 10–12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The blots were probed with primary antibodies against VEGFC or VEGFR3 (Abcam, Cambridge, UK). HRP-conjugated anti-rabbit or anti-rat IgG (Southern Biotech, Birmingham, AL) were used as the secondary antibodies.

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Quantifying LEC and ASC Signaling Dynamics

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Human dermal LECs and adipose-derived mesenchymal stem cells (ASCs) were obtained from PromoCell (Heidelberg, Germany) and stained in chamber slides for Prox1, Ki67 (Abcam), p-AKT (Cell Signaling Technology, Danvers, MA, USA) and VEGFR-3 (EMD Millipore, Billerica, MA, USA). Cell signal intensity was quantified using MetaMorph (Molecular Devices, Sunnyvale, CA, USA). Western blot analysis for VEGFR-3 (Abcam), p-AKT and p-eNOS (Cell Signaling Technology) was performed with total cellular protein harvested from LECs and quantified with NIH Image J.^{19 (link)}

García Nores G.D., Cuzzone D.A., Albano N.J., Hespe G.E., Kataru R.P., Torrisi J.S., Gardenier J.C., Savetsky I.L., Aschen S.Z., Nitti M.D, & Mehrara B.J. (2016). Obesity but not high-fat diet impairs lymphatic function. International Journal of Obesity (2005), 40(10), 1582-1590.

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Oleuropein-Mediated Angiogenesis Regulation

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Oleuropein (purity = 98.0%) was purchased from Extrasynthese (Genay, France). Antibodies against caveolin-1, F4/80, GLUT-1, Ki67, LYVE-1, VE-cadherin, VEGF-D and VEGFR3 were obtained from Abcam (Cambridge, MA, UK). HIF-1α, VEGF-A, VEGF-C, MMR, CDK4, cyclin D1 and CD31 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cleaved PARP and VEGFR2 antibodies were obtained from Cell Signaling (Beverly, MA, USA). Matrigel was purchased from Corning (MA, USA).

Song H., Lim D.Y., Jung J.I., Cho H.J., Park S.Y., Kwon G.T., Kang Y.H., Lee K.W., Choi M.S, & Park J.H. (2017). Dietary oleuropein inhibits tumor angiogenesis and lymphangiogenesis in the B16F10 melanoma allograft model: a mechanism for the suppression of high-fat diet-induced solid tumor growth and lymph node metastasis. Oncotarget, 8(19), 32027-32042.

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VEGFR2 and VEGFR3 Expression in Rat Lung

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Total protein was isolated from flash-frozen rat lung tissues using radio
immunoprecipitation assay lysis buffer followed by Western Blot for VEGFR2 (Cell
Signaling Technology, USA) and VEGFR3 (Abcam, USA) expression. The NIH ImageJ
software was used for densitometry analysis of immunoblots.

Agarwal S., Harter Z.J., Krishnamachary B., Chen L., Nguyen T., Voelkel N.F, & Dhillon N.K. (2020). Sugen–morphine model of pulmonary arterial hypertension. Pulmonary Circulation, 10(1), 2045894019898376.

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Anlotinib Inhibits Lymphangiogenesis

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Anlotinib was a gift from Jiangsu Chia-Tai Tianqing Pharmaceutical Co., Ltd. Antibodies against D2-40, podoplanin, LYVE-1, VEGFR-3, and Ki67 were purchased from Abcam (Cambridge, London, UK). Antibodies against p-Akt, Akt, p-Erk, Erk, VEGFR-2, p-VEGFR-2, and β-actin, as well as HRP-conjugated secondary antibodies were from CST (Danvers, MA), and antibody against p-VEGFR-3 (Tyr1230/1231) was from Cell Applications Inc. Alexa Fluor 555-conjugated secondary antibody was from Invitrogen Corporation (Carlsbad, CA). Freund’s incomplete adjuvant (IFA) was from Sigma-Aldrich (St Louis, MO). Human VEGF-C, a cytokine, was purchased from R&D Systems (Minneapolis, MN).

Qin T., Liu Z., Wang J., Xia J., Liu S., Jia Y., Liu H, & Li K. (2020). Anlotinib suppresses lymphangiogenesis and lymphatic metastasis in lung adenocarcinoma through a process potentially involving VEGFR-3 signaling. Cancer Biology & Medicine, 17(3), 753-767.

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Quantifying Lymphatic Endothelial Cell Signaling

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Human dermal LECs and adipose derived mesenchymal stem cells (ASCs) were obtained from PromoCell (Heidelberg, Germany) and stained in chamber slides for Prox1, Ki67 (Abcam, Cambridge, MA), p-AKT (Cell Signaling Technology, Danvers, MA), and VEGFR-3 (EMD Millipore, Billerica, MA). Cell signal intensity was quantified using MetaMorph (Molecular Devices, Sunnyvale, CA). Western blot analysis for VEGFR-3 (Abcam), p-AKT, and p-eNOS (Cell-signaling Technology) was performed with total cellular protein harvested from LECs and quantified with NIH Image J.(19 (link))

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Integrin-Mediated Adhesion Blockade

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The following antibodies were used for adhesion blockade: for integrin α₅ subunit (clone HMα5–1, Santa Cruz Biotechnology, Santa Cruz, CA), for integrin α_v subunit (clone H9.2B8, Santa Cruz Biotechnology), and for integrin α₉ subunit (antibody was supplied by Dr. Yasuyuki Yokosaki and generated in a similar manner as previously described) [24 (link)]. For immunostaining, antibodies used were against extra domain A (EIIIA) (clone IST9; Santa Cruz Biotechnology) (1:200), total fibronectin (Santa Cruz Biotechnology) (1:200), Ki-67 (Abcam) (1:200) and VE cadherin (R & D Systems, Minneapolis, MN) (1:200). For immunoblotting, antibodies used were against GAPDH (Abcam, Cambridge, MA) (1:10,000), total fibronectin (Santa Cruz Biotechnology) (1:1000), LYVE1 (Abcam)(1:100) and VEGFR3 (Abcam)(1:100).

Sackey-Aboagye B., Olsen A.L., Mukherjee S.M., Ventriglia A., Yokosaki Y., Greenbaum L.E., Lee G.Y., Naga H, & Wells R.G. (2016). Fibronectin Extra Domain A Promotes Liver Sinusoid Repair following Hepatectomy. PLoS ONE, 11(10), e0163737.

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