Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy

M Palmieri; CJ Nowell; M Condron; J Gardiner; AB Holmes; J Desai; AW Burgess; B Catimel

Journal article

Analysis of cellular phosphatidylinositol (3,4,5)-trisphosphate levels and distribution using confocal fluorescent microscopy

M Palmieri, CJ Nowell, M Condron, J Gardiner, AB Holmes, J Desai, AW Burgess, B Catimel

Analytical Biochemistry | ACADEMIC PRESS INC ELSEVIER SCIENCE | Published : 2010

DOI: 10.1016/j.ab.2010.06.033

Abstract

We have developed an immunocytochemistry method for the semiquantitative detection of phosphatidylinositol (3. ,4. ,5)-trisphosphate (PI(3,4,5)P3) at the cell plasma membrane. This protocol combines the use of a glutathione S-transferase-tagged pleckstrin homology (PH) domain of the general phosphoinositides-1 receptor (GST-GRP1PH) with fluorescence confocal microscopy and image segmentation using cell mask software analysis. This methodology allows the analysis of PI(3,4,5)P3 subcellular distribution in resting and epidermal growth factor (EGF)-stimulated HEK293T cells and in LIM1215 (wild-type phosphoinositide 3-kinase (PI3K)) and LIM2550 (H1047R mutation in PI3K catalytic domain) colonic ..

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University of Melbourne Researchers

Michelle Palmieri Author

Andrew Holmes Author

Jayesh Desai Author

Antony Burgess Author

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Grants

Awarded by Australian Research Council

Funding Acknowledgements

This work was supported by the Australian Research Council, Discovery Project (Grants DP0770668 and DP1094497), and the National Health and Medical Research Council (NHMRC) Program (Grant 487922). General support was provided by the Commonwealth Scientific and Industrial. Research Organisation (CSIRO) and the Victorian Endowment for Science, Knowledge, and Innovation (VESKI).