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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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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