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Purification of LAT-Containing Membranes from Resting and Activated T Lymphocytes

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The Immune Synapse

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1584))

Abstract

In T lymphocytes, the immune synapse is an active zone of vesicular traffic. Directional transport of vesicular receptors and signaling molecules from or to the immune synapse has been shown to play an important role in T-cell receptor (TCR) signal transduction. However, how vesicular trafficking is regulating the activation of T cells is still a burning question, and the characterization of these intracellular compartments remains the first step to understand this process. We describe herein a protocol, which combines a separation of membranes on flotation gradient with an affinity purification of Strep-tagged fusion transmembrane proteins with Strep-Tactin® resin, allowing the purification of membranes containing the Strep-tagged molecule of interest. By keeping the membranes intact, this protocol leads to the purification of molecules physically associated with the Strep-tagged protein as well as of molecules present in the same membrane compartment: transmembrane proteins, proteins strongly associated with the membranes, and luminal proteins. The example shown herein is the purification of membrane compartment prepared from T lymphocytes expressing LAT fused to a Strep-tag.

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Acknowledgments

We would like to thank R. Roncagalli for providing the LAT-Strep-tag® construct and for helpful discussion. We are grateful to S. Dogniaux, J.M. Carpier, M. Saitakis, and A. Zucchetti for their kind support.This work was supported by ANR-10-IDEX-0001-02 PSL*, ANR-11-LABX-0043, ANR-13-BSV2-0018 “NeuroImmunoSynapse”), and Fondation pour la Recherche Médicale (FRM, FRM DEQ20140329513).

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Authors and Affiliations

  1. Section Recherche, Institut Curie, INSERM U932, 26 Rue d’Ulm, 75248, Paris, Cedex 05, France

    Claire Hivroz, Mabel Jouve & Laurence Ardouin

  2. PSL Research University, Paris, France

    Claire Hivroz, Mabel Jouve & Laurence Ardouin

  3. Immunity and Cancer, INSERM U932, Paris, France

    Claire Hivroz, Mabel Jouve & Laurence Ardouin

  4. University of Milan, Milan, Italy

    Paola Larghi

  5. Istituto Nazionale Genetica Molecolare ‘Romeo ed Enrica Invernizzi’, INGM, Milan, Italy

    Paola Larghi

Authors
  1. Claire Hivroz
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  2. Paola Larghi
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  3. Mabel Jouve
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  4. Laurence Ardouin
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Correspondence to Laurence Ardouin .

Editor information

Editors and Affiliations

  1. Department of Life sciences, University of Siena, Siena, Siena, Italy

    Cosima T. Baldari

  2. University of Oxford, Kennedy Institute of Rheumatology, Headington, Oxford, United Kingdom

    Michael L. Dustin

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Hivroz, C., Larghi, P., Jouve, M., Ardouin, L. (2017). Purification of LAT-Containing Membranes from Resting and Activated T Lymphocytes. In: Baldari, C., Dustin, M. (eds) The Immune Synapse. Methods in Molecular Biology, vol 1584. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6881-7_21

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  • DOI: https://doi.org/10.1007/978-1-4939-6881-7_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6879-4

  • Online ISBN: 978-1-4939-6881-7

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