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In Situ Proximity Ligation Assay (In Situ PLA) to Assess PTP-Protein Interactions

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Protein Tyrosine Phosphatases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1447))

Abstract

Spatiotemporal aspects of protein-tyrosine phosphatase (PTP) activity and interaction partners for many PTPs are elusive. We describe here an elegant and relatively simple method, in situ proximity ligation assay (in situ PLA), which can be used to address these issues. The possibility to detect endogenous unmodified proteins in situ and to visualize individual interactions with spatial resolution is the major advantage of this technique. We provide protocols suitable to monitor association of the transmembrane PTPs PTPRJ/DEP-1/CD148 and PTPRB/VE-PTP with their substrates, the receptor tyrosine kinases FMS-like tyrosine kinase 3 (FLT3/CD135), and Tie2 and vascular endothelial growth factor receptor 2 (VEGFR2), respectively. Detailed description of method development and reagents as well as highlighting of critical factors will enable the reader to apply the method successfully to other PTP-protein interactions.

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Acknowledgments

The authors acknowledge grant support from Deutsche Forschungsgemeinschaft and Deutsche Krebshilfe (to F.D.B.), the Community’s 7th Framework Program (FP7/2007–2013) under grant agreement no 278568 “PRIMES” and the Swedish Research Council (to O.S.), the Knut and Alice Wallenberg foundation (Wallenberg Scholar Award), the Swedish Science Council, and the Swedish Cancer Foundation (to L.C-.W.).

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    Authors and Affiliations

    1. Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden

      Sina Koch, Irene Helbing, Makoto Hayashi, Lena Claesson-Welsh & Ola Söderberg

    2. Department 2, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany

      Sina Koch

    3. Alere Technologies GmbH, Jena, Germany

      Irene Helbing

    4. Institute of Molecular Cell Biology, CMB, Jena University Hospital, Hans-Knöll-Strasse 2, 07745, Jena, Germany

      Sylvia-Annette Böhmer & Frank-D. Böhmer

    5. Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe, Hyogo, Japan

      Makoto Hayashi

    6. Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical Center, Uppsala University, Uppsala, Sweden

      Ola Söderberg

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    1. Sina Koch
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    2. Irene Helbing
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    3. Sylvia-Annette Böhmer
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    6. Ola Söderberg
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    Correspondence to Frank-D. Böhmer .

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    Editors and Affiliations

    1. BioCruces Health Research Institute, Barakaldo, Bizkaia, Spain

      Rafael Pulido

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    Koch, S. et al. (2016). In Situ Proximity Ligation Assay (In Situ PLA) to Assess PTP-Protein Interactions. In: Pulido, R. (eds) Protein Tyrosine Phosphatases. Methods in Molecular Biology, vol 1447. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3746-2_13

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    • DOI: https://doi.org/10.1007/978-1-4939-3746-2_13

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    • Publisher Name: Humana Press, New York, NY

    • Print ISBN: 978-1-4939-3744-8

    • Online ISBN: 978-1-4939-3746-2

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