title: Identification of Connexin43 Phosphorylation and S-Nitrosylation in Cultured Primary Vascular Cells.
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.
authors.affiliation: Department of Cell Biology and Anatomy, Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada, T2N 4N1.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Lohman
authors.affiliation: Department of Pharmacology and Chemical Biology, Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Adam C
authors.initials: AC
authors.lastname: Straub
authors.affiliation: Institute of Cardiovascular and Medical Sciences, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK. scott.johnstone@glasgow.ac.uk.|BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA, UK. scott.johnstone@glasgow.ac.uk.
authors.forename: Scott R
authors.initials: SR
authors.lastname: Johnstone
mesh_terms: D001710:Biotin / Q000737:chemistry; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D018031:Connexin 43 / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000295:instrumentation / Q000379:methods; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D061251:Primary Cell Culture; D011499:Protein Processing, Post-Translational; D026403:S-Nitrosothiols / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
chemical_list: D018031:Connexin 43; C500216:GJA1 protein, human; D026403:S-Nitrosothiols; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D001710:Biotin; D003545:Cysteine
keywords: Biotin switch; Fluorescence-based Western blotting; Membrane preparation; Phosphorylation; Posttranslational modification; S-nitrosylation