RV infection induces release of larger quantities of proteins associated with MV. (A) Supernatants collected from RV-infected cells (MOI 5) or mock-infected Caco-2 cells for 24 h were filtered with 0.22-μm filters (F1), and ultracentrifuged at 100,000 × g for 90 min. The supernatant (F2) and pellet (F3) were recovered after ultracentrifugation. HSC70, HSP70, VP6, the ER protein calnexin, the exosome marker CD63, and lactadherin (MFG-E8, showing a 30-kDa intracellular protein form, and one 46 kDa in size associated with MV fractions) were evaluated in all fractions by Western blot. As a positive control we used 20 μg of cell lysate (Cx+), and equal volumes of each fraction were loaded in the gels. A representative Western blot of five independent experiments with similar results is shown. Western blots for CD63 identification were done in non-reducing conditions, producing a smeared band. (B) TGF-β1 was measured in five independent F3 preparations by ELISA. In three of these preparations AChE was simultaneously measured. The quantity of TGF-β1 is reported relative to the quantity of producing cells (p = 0.062 by Wilcoxon test for the comparison between mock- and RRV-infected F3), or to the AChE activity. The bars represent medians. (C) AChE activity was measured at 0, 6, 16, 24 and 48 h post-infection in apical and basolateral supernatants of RRV- and mock-infected Caco-2 cells, and the means from three independent experiments are shown.