A Monoclonal Antibody to Visualize PtdIns(3,4,5)P₃in Cells

Abstract

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.

Cross-Talk between CD14 and Complement Receptor 3 Promotes Phagocytosis of Mycobacteria: Regulation by Phosphatidylinositol 3-Kinase and Cytohesin-1

“…Cells were then permeabilized and stained with mAb RC6F8 followed by FITC-conjugated secondary Ab. The RC6F8 mAb recognized specifically PtdIns-3,4,5-P 3 , with minimal crossreactivity with PtdIns-(4,5)-diphosphate and for the two monophosphates PtdIns-(3)-monophosphate and PtdIns-(4)-monophosphate (35). The results in Fig.…”

Signal propagation from membrane messengers to nuclear effectors revealed by reporters of phosphoinositide dynamics and Akt activity

“…Biochemical methods of radiolabeling and cell fractionation measure total PI levels and have poor spatial and temporal resolution, and the use of specific antibodies (31) to immunodetect PIs in fixed cells provides only snapshots at different time points. For dynamic monitoring of the lipid molecules, live-cell imaging using PH domains fused with fluorescent proteins (10-13, 32, 33) has been widely used; however, their dependency on translocation limits their abilities to examine different pools of PIs.…”

Altered Insulin Signaling in Retinal Tissue in Diabetic States

“…This immunohistochemical approach has also proved useful for insulin signaling in other tissues including brain (32)(33)(34). In retina, PIP 3 is increased upon insulin stimulation only in endothelial cells that can be visualized by co-staining with the vascular marker, lectin B-4.…”