Fearnley, GW, Smith, GA, Odell, AF et al. (5 more authors) (2014) Vascular endothelial growth factor A-stimulated signaling from endosomes in primary endothelial cells. Methods in Enzymology, 535. 265 - 292. ISSN 0076-6879
Abstract
The vascular endothelial growth factor A (VEGF-A) is a multifunctional cytokine that stimulates blood vessel sprouting, vascular repair, and regeneration. VEGF-A binds to VEGF receptor tyrosine kinases (VEGFRs) and stimulates intracellular signaling leading to changes in vascular physiology. An important aspect of this phenomenon is the spatiotemporal coordination of VEGFR trafficking and intracellular signaling to ensure that VEGFR residence in different organelles is linked to downstream cellular outputs. Here, we describe a series of assays to evaluate the effects of VEGF-A-stimulated intracellular signaling from intracellular compartments such as the endosome-lysosome system. These assays include the initial isolation and characterization of primary human endothelial cells, performing reverse genetics for analyzing protein function; methods used to study receptor trafficking, signaling, and proteolysis; and assays used to measure changes in cell migration, proliferation, and tubulogenesis. Each of these assays has been exemplified with studies performed in our laboratories. In conclusion, we describe necessary techniques for studying the role of VEGF-A in endothelial cell function.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | (c) 2014, Elsevier. NOTICE: this is the author’s version of a work that was accepted for publication in Methods in Enzymology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Methods in Enzymology, 535, 2014. http://dx.doi.org/10.1016/B978-0-12-397925-4.00016... |
Keywords: | Endosome; Endothelial; GTPase; Migration; Proliferation; Signaling; Trafficking; Tubulogenesis; VEGF-A; VEGFR2 |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) |
Depositing User: | Symplectic Publications |
Date Deposited: | 19 Mar 2014 12:31 |
Last Modified: | 17 Aug 2015 13:36 |
Published Version: | http://dx.doi.org/10.1016/B978-0-12-397925-4.00016... |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/B978-0-12-397925-4.00016-X |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:78163 |