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Analysis of Aggrecanase Activity Using Neoepitope Antibodies

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ADAMTS Proteases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2043))

Abstract

Aggrecan is a major matrix component of articular cartilage, and its dysregulated proteolysis is a crucial event in the pathogenesis of arthritis. Aggrecanases, members of ADAMTS family, play a pivotal role in aggrecan degradation with ADAMTS-4 and ADAMTS-5 being key enzymes. Cleavage events mediated by ADAMTSs are highly specific and very well characterized; therefore, it is possible to investigate aggrecanolysis by using antibodies reacting with the new N- and C-termini of the cleavage products (neoepitope antibodies). Here, we present a method for analyzing dynamic aggrecanolysis by Western blotting using neoepitope antibodies in combination with antibodies against total aggrecan fragments. The protocol is robust and has a broad application for investigation of aggrecanase activity in vitro and ex vivo.

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Acknowledgments

SS is supported by the Wellcome Trust Institutional Strategic Support Fund’s Faculty Fellowship Scheme (ISSF) (Imperial College London). KY is supported by the Versus Arthritis UK Career Development Fellowship Scheme (21447). The authors thank Dr. Prof. Hideaki Nagase for his critical reading of the manuscript.

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Authors and Affiliations

  1. Centre for Haematology, Imperial College London, London, UK

    Salvatore Santamaria

  2. Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK

    Kazuhiro Yamamoto

Authors
  1. Salvatore Santamaria
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  2. Kazuhiro Yamamoto
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Corresponding author

Correspondence to Kazuhiro Yamamoto .

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Editors and Affiliations

  1. Department of Biomedical Engineering-ND20, Cleveland Clinic Lerner Research Institute, Cleveland, OH, USA

    Suneel S. Apte

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Santamaria, S., Yamamoto, K. (2020). Analysis of Aggrecanase Activity Using Neoepitope Antibodies. In: Apte, S. (eds) ADAMTS Proteases. Methods in Molecular Biology, vol 2043. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9698-8_11

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  • DOI: https://doi.org/10.1007/978-1-4939-9698-8_11

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9697-1

  • Online ISBN: 978-1-4939-9698-8

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