Identification of Connexin43 phosphorylation and S-nitrosylation in cultured primary vascular cells

Lohman, A. W., Straub, A. C. and Johnstone, S. R. (2016) Identification of Connexin43 phosphorylation and S-nitrosylation in cultured primary vascular cells. In: Vinken, M. and Johnstone, S. R. (eds.) Gap Junction Protocols. Series: Methods in molecular biology, 1437 (1437). Springer New York, pp. 97-111. ISBN 9781493936625 (doi: 10.1007/978-1-4939-3664-9_7)

Abstract

All connexins (Cx) proteins contain both highly ordered domains (i.e., 4 transmembrane domains) and primarily unstructured regions (i.e., n- and c-terminal domains). The c-terminal domains vary in length and amino acid composition from the shortest on Cx26 to the longest on Cx43. With the exception of Cx26, the c-terminal domains contain multiple sites for posttranslational modification (PTM) including serines (S), threonines (T), and tyrosines (Y) for phosphorylation or cysteines (C) for S-nitrosylation. These PTMs are critical for regulating cellular localization, protein–protein interactions, and channel functionality. There are several biochemical techniques that allow for the identification of these PTM including Western blotting and the “Biotin Switch” assay for nitrosylation. Quantitative analysis of Western blots can be achieved through use of secondary antibodies in the near infrared scale and high-resolution scanning on a fluorescent scanner.

• Item Type: Book Sections
• Additional Information: This work was financially supported by the University of Glasgow, Lord Kelvin Adam Smith Fellowship (SRJ), by NIH grants R00 HL11290402, the Institute for Transfusion Medicine and the Hemophilia Center of Western Pennsylvania (ACS) and postdoctoral fellowships from the Canadian Institutes for Health Research and Alberta Innovates - Health Solutions (AWL).
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Johnstone, Dr Scott
• Authors: Lohman, A. W., Straub, A. C., and Johnstone, S. R.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: Methods in Molecular Biology
• Publisher: Springer New York
• ISSN: 1064-3745
• ISSN (Online): 1940-6029
• ISBN: 9781493936625