DOI: 10.1177/002215540205000511. A Monoclonal Antibody to Visualize PtdIns(3,4,5)P<sub>3</sub> in Cells

A Monoclonal Antibody to Visualize PtdIns(3,4,5)P3 in Cells

10.1177/002215540205000511

Crossref journal-article

SAGE Publications

Journal of Histochemistry & Cytochemistry (179)

Abstract

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsPn) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsPn quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsPns. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P3 IgM, and immunodetection of PtdIns(3,4,5)P3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P3 relative to its precursor, phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P2), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was bench-marked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsPns. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P3 and PtdIns(4,5)P2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.

Bibliography

Chen, R., Kang, V. H., Chen, J., Shope, J. C., Torabinejad, J., DeWald, D. B., & Prestwich, G. D. (2002). A Monoclonal Antibody to Visualize PtdIns(3,4,5)P3 in Cells. Journal of Histochemistry & Cytochemistry, 50(5), 697â708.

Authors 7

Riyan Chen (first)
Veronica H. Kang (additional)
Jian Chen (additional)
Joseph C. Shope (additional)
Javad Torabinejad (additional)
Daryll B. DeWald (additional)
Glenn D. Prestwich (additional)

References 64 Referenced 59

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Dates

Type	When
Created	14 years, 4 months ago (April 1, 2011, 3:17 a.m.)
Deposited	5 months, 2 weeks ago (March 4, 2025, 11:26 a.m.)
Indexed	4 months, 3 weeks ago (March 30, 2025, 3:33 a.m.)
Issued	23 years, 3 months ago (May 1, 2002)
Published	23 years, 3 months ago (May 1, 2002)
Published Online	23 years, 3 months ago (May 1, 2002)
Published Print	23 years, 3 months ago (May 1, 2002)

Funders 0

None

BibTeX

@article{Chen_2002, title={A Monoclonal Antibody to Visualize PtdIns(3,4,5)P3 in Cells}, volume={50}, ISSN={1551-5044}, url={http://dx.doi.org/10.1177/002215540205000511}, DOI={10.1177/002215540205000511}, number={5}, journal={Journal of Histochemistry & Cytochemistry}, publisher={SAGE Publications}, author={Chen, Riyan and Kang, Veronica H. and Chen, Jian and Shope, Joseph C. and Torabinejad, Javad and DeWald, Daryll B. and Prestwich, Glenn D.}, year={2002}, month=may, pages={697–708} }

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  "abstract": "<jats:p> Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P<jats:sub>3</jats:sub>] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsP<jats:sub>n</jats:sub>) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsP<jats:sub>n</jats:sub> quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsP<jats:sub>n</jats:sub>s. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P<jats:sub>3</jats:sub> immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P<jats:sub>3</jats:sub> IgM, and immunodetection of PtdIns(3,4,5)P<jats:sub>3</jats:sub> in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P<jats:sub>3</jats:sub> relative to its precursor, phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P<jats:sub>2</jats:sub>), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was bench-marked against HPLC analysis of [<jats:sup>3</jats:sup>H]-myo-inositol-labeled cellular PtdInsP<jats:sub>n</jats:sub>s. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P<jats:sub>3</jats:sub> and PtdIns(4,5)P<jats:sub>2</jats:sub> in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P<jats:sub>3</jats:sub> levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P<jats:sub>2</jats:sub> levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase. </jats:p>",
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