Abstract
This chapter describes an indirect approach to measure PTEN’s lipid phosphatase activity in vivo. PTEN counteracts phosphatidylinositol 3-kinase action in dephosphorylating 3-phosphorylated phosphoinositides. Therefore, PtdIns(3,4,5)P3-dependent activation and phosphorylation of the survival kinase Akt can be used as readout for cellular PTEN activity. Here we have outlined a detailed procedure employing a phosphoserine-specific anti-Akt antibody to examine the content of phosphorylated Akt by immunofluorescence and its dependence on PTEN activity.
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Abbreviations
- BSA::
-
bovine serum albumin;
- PtdIns(3,4,5)P 3::
-
phosphatidylinositol 3,4,5-trisphosphate;
- PtdIns(3,4)P2::
-
phosphatidylinositol 3,4-bisphosphate;
- PTEN::
-
phosphatase and tensin homologue
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Acknowledgments
We thank Peter Parker (Cancer Research UK) for providing the phospho-peptide. This work was supported by MRC and BBSRC.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Rosivatz, E., Woscholski, R. (2009). Measurement of PTEN Activity in vivo by Imaging Phosphorylated Akt. 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_14
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DOI: https://doi.org/10.1007/978-1-60327-115-8_14
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