Open AccessA Monoclonal Antibody to Visualize PtdIns(3,4,5)P3in Cells
Author(s) -
Riyan Chen,
Veronica H. Kang,
Jian Chen,
Joseph C. Shope,
Javad Torabinejad,
Daryll B. DeWald,
Glenn D. Prestwich
Publication year - 2002
Publication title -
journal of histochemistry and cytochemistry/the journal of histochemistry and cytochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.971
H-Index - 124
eISSN - 1551-5044
pISSN - 0022-1554
DOI - 10.1177/002215540205000511
Subject(s) - monoclonal antibody , antibody , chemistry , microbiology and biotechnology , computational biology , medicine , immunology , biology
Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsP n ) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsP n quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsP n s. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P 3 immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P 3 IgM, and immunodetection of PtdIns(3,4,5)P 3 in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P 3 relative to its precursor, phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P 2 ), 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 [ 3 H]-myo-inositol-labeled cellular PtdInsP n s. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P 3 and PtdIns(4,5)P 2 in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P 3 levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P 2 levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.