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Differential Detection of O-GlcNAcylated proteins in the heart using antibodies.
Narayanan, Bhargavi; Zahra, Fiddia; Reeves, Russell A; Aggarwal, Akanksha; O'Meally, Robert N; Henry, Roger K; Craven, Megan; Jacobson, Avital; Cole, Robert N; Kohr, Mark J; Umapathi, Priya; Zachara, Natasha E.
Afiliación
  • Narayanan B; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • Zahra F; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • Reeves RA; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; Department of Radiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, 90095, USA.
  • Aggarwal A; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • O'Meally RN; The Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • Henry RK; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; Department of Ophthalmology and Visual Sciences at the Rutgers University New Jersey Medical School Newark, NJ, 07103, USA.
  • Craven M; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • Jacobson A; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • Cole RN; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; The Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • Kohr MJ; The Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, 21205, USA.
  • Umapathi P; The Department Medicine at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
  • Zachara NE; The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; The Department of Oncology at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA. Electronic address: nzachara@jhmi.edu.
Anal Biochem ; 678: 115262, 2023 10 01.
Article en En | MEDLINE | ID: mdl-37507081
ABSTRACT
Thousands of mammalian intracellular proteins are dynamically modified by O-linked ß-N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Enfermedades Neurodegenerativas Tipo de estudio: Diagnostic_studies Límite: Animals Idioma: En Revista: Anal Biochem Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Enfermedades Neurodegenerativas Tipo de estudio: Diagnostic_studies Límite: Animals Idioma: En Revista: Anal Biochem Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos