FIG 2.

Identification of vesicular markers in vesicles purified from Caco-2 cells. Caco-2 cells were infected or mock-infected with astrovirus strain Yuc8 (MOI of 5) and 18 h postinfection the supernatant was collected. Different fractions were purified by differential centrifugation; at 500 g pellet 1 was obtained (P1), pellet 2 was obtained at 2,000 g (P2), large extracellular vesicles (LEV) were obtained after 20,000 g centrifugation and final small extracellular vesicles (SEV) fraction was obtained by precipitation with 8% polyethylene glycol 6000, 0.5M NaCl. (A) The same volume of each pelleted fraction was separated by SDS-PAGE and proteins were detected by silver staining. (B) Samples were resolved on SDS-PAGE and analyzed by Western blotting, using antibodies specific for CD63 and Alix as vesicle markers and protein disulfide isomerase (PDI) as endoplasmic reticulum protein to assess preparation contamination. To detect GM1 (general membrane marker) samples were directly blotted onto nitrocellulose membrane and processed. Immunoblots are representative of five independent experiments.