Abstract
The seven phosphorylated derivatives of phosphatidylinositol (PtdIns), often collectively referred to as polyphosphoinositides (PPIn), are a minor component of eukaryotic cell membranes. Nevertheless, their synthesis is needed for an ever-increasing spectrum of cellular processes, including regulation of the actin cytoskeleton, chemotaxis, membrane trafficking, glucose uptake, and organelle acidification. PPIn metabolism is regulated dynamically by a network of kinases and phosphatases. Furthermore, synthesis of PPIn can be provoked by external stimuli; for example, the second messenger phosphatidylinositol 3,4,5-trisphosphate rapidly and transiently accumulates in cells challenged with agonists such as PDGF that activate receptor tyrosine kinases. The measurement of PPIn levels in in vivo cultured cells has been vital to our understanding of the metabolism and function of these important signaling molecules; methods are described herein that allow measurement of PPIn levels in culture cells in vivo.
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Abbreviations
- BSA::
-
bovine serum albumin;
- cpm::
-
counts per minute;
- dpm::
-
disintegrations per minute;
- FAF::
-
fatty acid-free;
- GroP::
-
glycerophospho;
- HEPES::
-
N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid;
- HBBSS::
-
buffered balanced salts solution;
- HPLC::
-
high-performance liquid chromatography;
- Ins::
-
inositol;
- Ptd::
-
phosphatidyl;
- PPIn::
-
polyphosphoinositide;
- Pi::
-
inorganic
- PO4::
- TBAS::
-
tetrabutyl ammonium hydrogen sulfate.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Cooke, F.T. (2009). Measurement of Polyphosphoinositides in Cultured Mammalian Cells. In: Larijani, B., Woscholski, R., Rosser, C. (eds) Lipid Signaling Protocols. Methods in Molecular Biology, vol 462. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-115-8_3
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DOI: https://doi.org/10.1007/978-1-60327-115-8_3
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-60327-115-8
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