Nef detection in the AChE+ fractions from Nef-transfected 293T.

293T (2 x 10⁶) were plated in a 10 cm plate and transfected with GFP (A-C), Nef.GFP (A, D & E), or CD81.GFP (F-H). Transfected cells were then cultured in exosome-free medium for 3 days. Culture medium was collected and pooled (about 70 ml total) for crude exosomes (500 μl) as described above, while cells were harvested for cell lysates for whole cell extracts (WCE). WCE and crude exosomes were analyzed by Western blotting using TSG101 and cytochrome C (cyto C) antibody (A). GFP and Nef.GFP and CD81.GFP were visualized at a wavelength of 488 nm (A & F). The data were representative of three independent experiments. Then the crude exosomes were subjected to the OptiPrep gradient centrifugation and fractionated. Aliquots of each fraction were used for AChE activity assay (24 μl, B, D & G). The remaining fractions were diluted in 4 ml PBS and spun at 100,000 g, 70 min. The pellets were lysed in the RIPA buffer followed by Western blotting using indicated antibodies (C, E & H). WCE (100 μg) were included as controls (C, E & H). The AChE activities were mean ± SD of triplicates; the data were representative of three independent experiments.