Intracellular Staining by Flow Cytometry
|Detection of Insulysin/IDE in HeLa cells by Flow Cytometry. HeLa cells were stained with Mouse Anti-Human Insulysin/IDE Monoclonal Antibody (Catalog # MAB2496, filled histogram) or isotype control antibody (Catalog # MAB002, open histogram), followed by Allophycocyanin-conjugated Anti-Mouse IgG F(ab')2 Secondary Antibody (Catalog # F0101B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
Insulysin, or insulin-degrading enzyme (IDE), is a zinc metallopeptidase of the inverzincin family. IDE is primarily located in the cytosol, but has been detected as a secreted enzyme and associated with the plasma membrane as well (1). The enzyme is expressed in many tissues, with the highest levels in liver, kidney, brain, and testis (2). IDE hydrolyzes a variety of regulatory peptides, including insulin, glucagon, atrial natriuretic factor, and transforming growth factor-alpha in vitro (1). In addition, IDE has been shown to degrade the amyloid beta (A beta ) peptide, which polymerizes into the plaques associated with Alzheimer's disease (3). Deficiencies in IDE activity may contribute to the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimer's disease. The IDE region of human chromosome 10q has been genetically linked to DM2 (4). When the IDE gene was specifically disrupted in mice, IDE -/- animals developed hyperinsulinemia and glucose intolerance, characteristics of DM2 (5). The IDE -/- mice were also shown to have a significant decrease in A beta degradation in the brain, resulting in increased cerebral accumulation of A beta peptide. This in vivo evidence is consistent with the hypotheses that IDE is important for the degradation of insulin in cells and for the clearance of A beta peptide in the brain.