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Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes

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Phosphoinositides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2251))

Abstract

Mammalian phospholipase C (PLC) isozymes are major signaling nodes that regulate a wide range of cellular processes. Dysregulation of PLC activity has been associated with a growing list of human diseases such as cancer and Alzheimer’s disease. However, methods to directly and continuously monitor PLC activity at membranes with high sensitivity and throughput are still lacking. We have developed XY-69, a fluorogenic PIP2 analog, which can be efficiently hydrolyzed by PLC isozymes either in solution or at membranes. Here, we describe the optimized assay conditions and protocol to measure the activity of PLC-γ1 (D1165H) with XY-69 in lipid vesicles. The described protocol also applies to other PLC isozymes.

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Acknowledgments

This work was supported by the National Institutes of Health (GM057391 to J. S. and CA177993 to Q. Z.) and the North Carolina Biotechnology Center (TEG-2018-1505 to Q. Z.). We thank Dr. Xiaoyang Wang for providing XY-69 and the Center for Integrative Chemical Biology and Drug Discovery (CICBDD) at the UNC Eshelman School of Pharmacy for the access to a microplate reader.

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Authors and Affiliations

  1. Division of Chemical Biology and Medicinal Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Adam J. Carr, Weigang Huang & Qisheng Zhang

  2. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Edhriz Siraliev-Perez & John Sondek

  3. Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    John Sondek & Qisheng Zhang

  4. The Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    John Sondek & Qisheng Zhang

Authors
  1. Adam J. Carr
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  2. Edhriz Siraliev-Perez
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  3. Weigang Huang
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  4. John Sondek
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  5. Qisheng Zhang
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Corresponding author

Correspondence to Qisheng Zhang .

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Editors and Affiliations

  1. Department of Chemistry and Biology and the Graduate Program in Molecular Science, Ryerson University, Toronto, ON, Canada

    Roberto J. Botelho

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Carr, A.J., Siraliev-Perez, E., Huang, W., Sondek, J., Zhang, Q. (2021). Fluorogenic XY-69 in Lipid Vesicles for Measuring Activity of Phospholipase C Isozymes. In: Botelho, R.J. (eds) Phosphoinositides. Methods in Molecular Biology, vol 2251. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1142-5_17

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  • DOI: https://doi.org/10.1007/978-1-0716-1142-5_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1141-8

  • Online ISBN: 978-1-0716-1142-5

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