S U M M A R Y Phosphatidylinositol 3,4,4,5)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-bisphosphate (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 benchmarked 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.
