Figure 3: Ago2 and miRNA are present within purified EVs.

(a) Expression of Ago2 and stomatin in multiple EV preparations was probed by western blot. (b) Immuno electron microscopy. EVs were prepared and processed as described in materials and methods for labelling with human Ago2 antibody. The field of view shows multiple EVs with internal immunogold labelling, demonstrating that Ago2 is present in EVs rather than associated by surface attachment. Inserts: three representative EVs in higher magnification (scale bar, 100 nm). (c) Elution profile of purified EVs, human serum, BSA and tyrosine (Tyr) after size-exclusion separation. Protein abundance was determined by absorbance at 280 nm. Points represent the mean±s.d. of three experiments performed in triplicate. (d) Fractions from purified EVs (left) and serum (right) were assayed for miR451a (black) and let-7a (red) using absolute quantification by TaqMan qPCR. The mean±s.d. of one representative experiment is shown (n=2). (e,f) The Ago2-miRNA complexes are protected from protease K digestion. (e) EVs were treated with proteinase K (5 mg ml⁻¹, or untreated control) at 55 °C. At times indicated, aliquots were removed and assessed for Ago2, stomatin and Glycophorin C expression by western Blot. In contrast to Glycophorin C, Ago2 and stomatin were protected from digestion by proteinase K. (f) Untreated (open symbols) or treated samples (close symbols) were also analysed for miR-451a and let-7a expression by qPCR. Points represent miRNA copies detected at each time relative to the control sample at time point 0. The mean±s.d. of one representative experiment is shown (n=3). qPCR, quantitative PCR.