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RNAi-Based Screening for the Identification of Specific Substrate-Deubiquitinase Pairs

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The Ubiquitin Code

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2602))

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Abstract

Ubiquitination signals are regulated in time and space due to the coordinated action of E3s and DUBs, which enables the precise control of cellular function and homeostasis. Mutations in all types of ubiquitin-proteasome system (UPS) components are related to pathological conditions. The identification of E3/DUBs’ ubiquitinated substrates can provide a clearer view of the molecular mechanisms underlying those diseases. However, the analysis of ubiquitinated proteins is not trivial. Here, we propose a protocol to identify DUB/substrate pairs, by combining DUB silencing, specific pull-down of the substrate, and image analysis of its ubiquitinated fraction.

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Acknowledgments

We thank Juanma Ramirez, Nerea Osinalde, and the rest of the group members for various contributions to the development of this protocol. We also thank Josean Rodriguez for providing valuable advice. This work was supported with MINECO SAF2016-76898-P and PID2020-117333GB-I00 grants, as well as with Basque Government GIC18/150 grant.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Leioa, Spain

    Nagore Elu, Natalia Presa & Ugo Mayor

  2. Ikerbasque, Basque Foundation for Science, Bilbao, Spain

    Ugo Mayor

Authors
  1. Nagore Elu
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  2. Natalia Presa
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  3. Ugo Mayor
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Corresponding author

Correspondence to Ugo Mayor .

Editor information

Editors and Affiliations

  1. LCC-UPR 8241-CNRS Equipe M, Laboratoire de Chimie de Coordination, Toulouse, France

    Manuel S Rodriguez

  2. CIC bioGUNE, Derio, Spain

    Rosa Barrio

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Elu, N., Presa, N., Mayor, U. (2023). RNAi-Based Screening for the Identification of Specific Substrate-Deubiquitinase Pairs. In: Rodriguez, M.S., Barrio, R. (eds) The Ubiquitin Code. Methods in Molecular Biology, vol 2602. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2859-1_7

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  • DOI: https://doi.org/10.1007/978-1-0716-2859-1_7

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2858-4

  • Online ISBN: 978-1-0716-2859-1

  • eBook Packages: Springer Protocols

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