Comparative analysis of antibodies to xCT (Slc7a11): Forewarned is forearmed

  • Joeri Van Liefferinge
    Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences Vrije Universiteit Brussel Brussels 1090 Belgium
  • Eduard Bentea
    Department of Pharmaceutical Biotechnology and Molecular Biology, Center for Neurosciences Vrije Universiteit Brussel Brussels 1090 Belgium
  • Thomas Demuyser
    Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences Vrije Universiteit Brussel Brussels 1090 Belgium
  • Giulia Albertini
    Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences Vrije Universiteit Brussel Brussels 1090 Belgium
  • Virginie Follin‐Arbelet
    Department of Molecular Medicine, Institute of Basic Medical Sciences University of Oslo Oslo 0317 Norway
  • Silvia Holmseth
    Department of Molecular Medicine, Institute of Basic Medical Sciences University of Oslo Oslo 0317 Norway
  • Ellen Merckx
    Department of Pharmaceutical Biotechnology and Molecular Biology, Center for Neurosciences Vrije Universiteit Brussel Brussels 1090 Belgium
  • Hideyo Sato
    Laboratory of Biochemistry and Molecular Biology, Department of Medical Technology Niigata University Niigata Niigata Prefecture 950‐2181 Japan
  • Joeri L. Aerts
    Laboratory of Molecular and Cellular Therapy, Department of Immunology–Physiology Vrije Universiteit Brussel Brussels 1090 Belgium
  • Ilse Smolders
    Department of Pharmaceutical Chemistry and Drug Analysis, Center for Neurosciences Vrije Universiteit Brussel Brussels 1090 Belgium
  • Lutgarde Arckens
    Laboratory of Neuroplasticity and Neuroproteomics KU Leuven Leuven 3000 Belgium
  • Niels C. Danbolt
    Department of Molecular Medicine, Institute of Basic Medical Sciences University of Oslo Oslo 0317 Norway
  • Ann Massie
    Department of Pharmaceutical Biotechnology and Molecular Biology, Center for Neurosciences Vrije Universiteit Brussel Brussels 1090 Belgium

説明

<jats:title>ABSTRACT</jats:title><jats:p>The cystine/glutamate antiporter or system <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/cne23889-math-0002.png" xlink:title="urn:x-wiley:00219967:media:cne23889:cne23889-math-0002" /> exchanges cystine for glutamate, thereby supporting intracellular glutathione synthesis and nonvesicular glutamate release. The role of system <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/cne23889-math-0003.png" xlink:title="urn:x-wiley:00219967:media:cne23889:cne23889-math-0003" /> in neurological disorders can be dual and remains a matter of debate. One important reason for the contradictory findings that have been reported to date is the use of nonspecific anti‐xCT (the specific subunit of system <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="graphic/cne23889-math-0004.png" xlink:title="urn:x-wiley:00219967:media:cne23889:cne23889-math-0004" />) antibodies. Often studies rely on the predicted molecular weight of 55.5 kDa to identify xCT on Western blots. However, using brain extracts from xCT knockout (xCT<jats:sup>−/−</jats:sup>) mice as negative controls, we show that xCT migrates as a 35‐kDa protein. Misinterpretation of immunoblots leads to incorrect assessment of antibody specificity and thereby to erroneous data interpretation. Here we have verified the specificity of most commonly used commercial and some in‐house‐developed anti‐xCT antibodies by comparing their immunoreactivity in brain tissue of xCT<jats:sup>+/+</jats:sup> and xCT<jats:sup>−/−</jats:sup> mice by Western blotting and immunohistochemistry. The Western blot screening results demonstrate that antibody specificity not only differs between batches produced by immunizing different rabbits with the same antigen but also between bleedings of the same rabbit. Moreover, distinct immunohistochemical protocols have been tested for all the anti‐xCT antibodies that were specific on Western blots in order to obtain a specific immunolabeling. Only one of our in‐house‐developed antibodies could reveal specific xCT labeling and exclusively on acetone‐postfixed cryosections. Using this approach, we observed xCT protein expression throughout the mouse forebrain, including cortex, striatum, hippocampus, midbrain, thalamus, and amygdala, with greatest expression in regions facing the cerebrospinal fluid and meninges. J. Comp. Neurol. 524:1015–1032, 2016. © 2015 Wiley Periodicals, Inc.</jats:p>

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