Fig. 1
From: Alix and Syntenin-1 direct amyloid precursor protein trafficking into extracellular vesicles
Amyloid precursor protein and amyloid beta are packaged into small extracellular vesicles. a Immunoblot analysis of HEK293 cell-derived EVs harvested by modified differential centrifugation following APPswe transfection. b Schematic of APP proteolytic processing and epitope binding by several commercial antibody clones targeting APP metabolites. c EV protein was titrated and probed by several antibodies recognizing the C-terminus of APP (A8717) or N-terminus of Aβ/CTFβ (6E10, 2454) in comparison to purified oligomerized Aβ. d EVs were enriched by polyethylene glycol incubation and ultracentrifugation before subsequent purification and fractionation on an iodixanol density gradient. Equal volume was loaded for immunoblot analysis. One μg of cell lysate was run to demonstrate depletion of Calnexin in isolated EV fractions. Blots are representative images from at least three repeated independent experiments. e Densities of gradient separated fractions were estimated by measuring refractive indices of fractions with a refractometer. f Electron microscopy of gradient purified EVs (scale bar = 200 nm), and g) nanoparticle tracking analysis of small EVs (100 K g pellet) secreted from control and APPswe transfected cells averaged across three biological replicates