Abstract
The chromatin immunoprecipitation (ChIP) assay is a versatile technique used to evaluate the association of proteins with specific DNA regions both in vivo and in vitro. This assay can be used to identify proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein. The ChIP assay can also be used to analyze binding of transcription factors, transcription cofactors, DNA replication factors, and DNA repair proteins. Here we describe a useful ChIP-qPCR protocol to examine the interaction of NFkB with the VEGF promoter in adult rat primary cardiomyocytes that have been mechanically stretched after attaching to the extracellular matrix protein laminin.
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References
Furey TS (2012) ChIP-seq and beyond: new and improved methodologies to detect and characterize protein-DNA interactions. Nat Rev Genet 13(12):840–852
Goehringer C, Rutschow D, Bauer R et al (2009) Prevention of cardiomyopathy in delta-sarcoglycan knockout mice after systemic transfer of targeted adeno-associated viral vectors. Cardiovasc Res 82(3):404–410
Leychenko A, Konorev E, Jijiwa M et al (2011) Stretch-induced hypertrophy activates NFkB-mediated VEGF secretion in adult cardiomyocytes. PLoS One 6(12), e29055
Sikder D, Kodadek T (2005) Genomic studies of transcription factor-DNA interactions. Curr Opin Chem Biol 9(1):38–45
Piper HM, Volz A, Schwartz P (1990) Adult ventricular rat heart muscle cells. In: Piper HM (ed) Cell culture techniques in heart and vessel research. Springer, Heidelberg, pp 36–60
Ramanathan M, Pinhal-Enfield G, Hao I et al (2007) Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter. Mol Biol Cell 18(1):14–23
Chiu KP, Wong C-H, Chen Q et al (2006) PET-Tool: a software suite for comprehensive processing and managing of Paired-End diTag (PET) sequence data. BMC Bioinformatics 7:390
Harlow E, Lane D (1988) Antibodies, A laboratory manual. Cold spring harbor laboratory, NY, 617–618
Acknowledgments
The authors thank Anna Leychenko for technical help with the ARCM isolation, stretch, and ChIP assay. M.L. Matter is supported by a grant from the National Institutes of Health (RO1GM104984) and C.B. Walton is supported by RR016453-09.
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Walton, C.B., Matter, M.L. (2015). Chromatin Immunoprecipitation Assay: Examining the Interaction of NFkB with the VEGF Promoter. In: Fiedler, L. (eds) VEGF Signaling. Methods in Molecular Biology, vol 1332. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2917-7_6
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DOI: https://doi.org/10.1007/978-1-4939-2917-7_6
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2916-0
Online ISBN: 978-1-4939-2917-7
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