Nef and CD81 localization in intracellular and extracellular vesicles.

<p>293T (5 X 10<sup>4</sup>) were plated in a 24-well plate and transfected with GFP, Nef.GFP, or CD81.GFP plasmid. Twenty-four hours post transfection, the cells were re-plated on top of polylysine-treated coverslip in a 24-well plate. Cells were fixed after 24 hr and processed for immunostaining using an anti-CD81 antibody, followed by Alexa Fluor 555-conjugated goat anti-mouse secondary antibody, which allows detection of both endogenous and exogenous CD81 expression and localization using a rhodamine filter under confocal microscope (60X objective). GFP tagged protein expression and localization were detected using a FITC filter. <b>A.</b> CD81 Nef co-localization; <b>B.</b> Extracellular vesicle-like structures (with extended exposure time). The micrographs were representative of images from multiple fields of three independent experiments.</p>