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LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks

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Book cover Proteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1550))

Abstract

Protein–protein interactions (PPIs) play an essential role in all biological processes. In vivo, PPIs occur dynamically and depend on extracellular cues. To discover novel protein–protein interactions in mammalian cells, we developed a high-throughput automated technology called LUMIER (LUminescence-based Mammalian IntERactome). In this approach, we co-express a Luciferase (LUC)-tagged fusion protein along with a Flag-tagged protein in an efficiently transfectable cell line such as HEK-293T cells. The interaction between the two proteins is determined by co-immunoprecipitation using an anti-Flag antibody, and the presence of the LUC-tagged interactor in the complex is subsequently detected via its luciferase activity. LUMIER can easily detect transmembrane protein partners, interactions that are signaling- or splice isoform-dependent, as well as those that may occur only in the presence of posttranslational modifications. Using various collections of Flag-tagged proteins, we have generated protein interaction networks for several TGF-β family receptors, Wnt pathway members, and have systematically analyzed the effect of neural-specific alternative splicing on protein interaction networks. The results have provided important insights into the physiological and functional relevance of some of the novel interactions found. LUMIER is highly scalable and can be used for both low- and high-throughput strategies. LUMIER is thus a valuable tool for the identification and characterization of dynamically regulated PPIs in mammalian systems. Here, we describe a manual version of LUMIER in a 96-well format that can be easily implemented in any laboratory.

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Acknowledgments

This work was supported by funding from the Canadian Institutes of Health Research (CIHR) (J.L.W. and B.J.B.), the Alzheimer’s Society of Canada (B.J.B.), and the Krembil Foundation (J.L.W.). We would also like to thank Dr. Saranya Kittanakom for the plasmid schematics framework.

Author information

Authors and Affiliations

  1. Center for Systems Biology, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON, Canada, M5G 1X5

    Miriam Barrios-Rodiles & Jeffrey L. Wrana Ph.D., F.R.S.C.

  2. Donnelly Centre, University of Toronto, Toronto, ON, Canada, M5S 3E1

    Jonathan D. Ellis & Benjamin J. Blencowe

  3. Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada

    Benjamin J. Blencowe & Jeffrey L. Wrana Ph.D., F.R.S.C.

  4. Breast Cancer Research, Mary Janigan Chair in Molecular Cancer Therapeutics, Toronto, ON, Canada

    Jeffrey L. Wrana Ph.D., F.R.S.C.

Authors
  1. Miriam Barrios-Rodiles
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  2. Jonathan D. Ellis
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  3. Benjamin J. Blencowe
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  4. Jeffrey L. Wrana Ph.D., F.R.S.C.
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Corresponding author

Correspondence to Miriam Barrios-Rodiles .

Editor information

Editors and Affiliations

  1. Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Lucio Comai

  2. Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Jonathan E. Katz

  3. Stanford University, Palo Alto, California, USA

    Parag Mallick

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Barrios-Rodiles, M., Ellis, J.D., Blencowe, B.J., Wrana, J.L. (2017). LUMIER: A Discovery Tool for Mammalian Protein Interaction Networks. In: Comai, L., Katz, J., Mallick, P. (eds) Proteomics. Methods in Molecular Biology, vol 1550. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6747-6_11

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  • DOI: https://doi.org/10.1007/978-1-4939-6747-6_11

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6745-2

  • Online ISBN: 978-1-4939-6747-6

  • eBook Packages: Springer Protocols

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