Protecting the secreted miRNAs that are stored in MVs, we further added proteinase K (PK) into the digestion system containing TX-100 and RNaseA. As shown, all of the miRNAs were now completely degraded (Figure 1D). Together, these results suggest that both the MVs and proteins contribute to the 4EGI-1 supplier resistance to RNase A of secreted miRNAs in the MVs. The protection by the MVs is non-specific, whereas the protection by proteins is selective for particular miRNAs. In agreement with the observation that the stability of secreted miRNAs is dependent on MVs and proteins, we found that all eight miRNAs, after extracted from the plasma, were completely degraded by RNaseA (Figure 1E), which is in agreement with the previous finding that the circulating miRNAs are generally not intrinsically resistant to RNases [19].Identification of Ago2 as a key protein protecting secreted miRNAs in MVsPrevious studies [11,27?9] showed that Ago2 and CD63 were located in MVs. Employing CD63 as an exosomal marker, we confirmed that the isolated MVs from cultured HeLa cells were enriched in both CD63 and Ago2 (Figure 2A). Because the miR16 in the MVs was strongly protected by a proteinase-sensitive mechanism (Figure 1D), we designed a miR-16 pull-down strategy to isolate potential miR-16-associated proteins using the MV fractions isolated from human plasma (Figure 2B). The pull-down product by the biotin-labeled probe complementary to human miR-16 (add adenosine at the 59 and 39 ends, respectively) 25837696 was further separated by SDS-PAGE followed by silver staining or by western blotting using anti-Ago2 and anti-CD63 antibodies in a parallel fashion. As shown in Figure 2C, although both Ago2 and CD63 were enriched in the MVs, only Ago2 was found to be associated with miR-16. We 
also employed the same strategy to isolate potential miR-223-associated proteins using the MV fractions K162 derived from human plasma (Figure S1). As can be seen, Ago2 was also identified as a major protein band associated with miR-223 though the amount of Ago2 associated with miR-223 was slightly less than that associated with miR-16. We then analyzed the association of various miRNAs with the Ago2 complexes in the MVs by immunoprecipitating Ago2 using an anti-Ago2 antibody, followed by the detection of the miRNAs using TaqMan probe-based qRT-PCR. Interestingly, we found that the association of the MV-encapsulated miRNAs with the Ago2 complexes was variable, and among the eight miRNAs that we tested, miR-16 showed the highest percentage of total miRNA to associate with the Ago2 complexes. Combining this observation with the result from Figure 1D, we speculate that the degree of association of miRNAs with the Ago2 complexes is positively correlated with the resistance of the miRNAs to degradation by RNaseA. To further analyze the protection provided by Ago2 complexes to miRNAs in the MVs, we compared the stability of mature miR16 that was associated with Ago2 complexes with that of free, synthetic, mature miR-16. In this experiment, the Ago2-associated miRNAs in the MVs, including miR-16, were harvested byimmunoprecipitating the lysate of MV fraction using an anti-Ago2 antibody, and the amount of Ago2-associated miR-16 in the precipitated product was quantitatively analyzed by qRT-PCR, referring to the standard curve of miR-16. An equal amount of Ago2-associated miR-16 and free, synthetic miR-16 were then treated with RNaseA at various concentrations and for 
various time periods. As shown in Figure 3A.Protecting the secreted miRNAs that are stored in MVs, we further added proteinase K (PK) into the digestion system containing TX-100 and RNaseA. As shown, all of the miRNAs were now completely degraded (Figure 1D). Together, these results suggest that both the MVs and proteins contribute to the resistance to RNase A of secreted miRNAs in the MVs. The protection by the MVs is non-specific, whereas the protection by proteins is selective for particular miRNAs. In agreement with the observation that the stability of secreted miRNAs is dependent on MVs and proteins, we found that all eight miRNAs, after extracted from the plasma, were completely degraded by RNaseA (Figure 1E), which is in agreement with the previous finding that the circulating miRNAs are generally not intrinsically resistant to RNases [19].Identification of Ago2 as a key protein protecting secreted miRNAs in MVsPrevious studies [11,27?9] showed that Ago2 and CD63 were located in MVs. Employing CD63 as an exosomal marker, we confirmed that the isolated MVs from cultured HeLa cells were enriched in both CD63 and Ago2 (Figure 2A). Because the miR16 in the MVs was strongly protected by a proteinase-sensitive mechanism (Figure 1D), we designed a miR-16 pull-down strategy to isolate potential miR-16-associated proteins using the MV fractions isolated from human plasma (Figure 2B). The pull-down product by the biotin-labeled probe complementary to human miR-16 (add adenosine at the 59 and 39 ends, respectively) 25837696 was further separated by SDS-PAGE followed by silver staining or by western blotting using anti-Ago2 and anti-CD63 antibodies in a parallel fashion. As shown in Figure 2C, although both Ago2 and CD63 were enriched in the MVs, only Ago2 was found to be associated with miR-16. We also employed the same strategy to isolate potential miR-223-associated proteins using the MV fractions derived from human plasma (Figure S1). As can be seen, Ago2 was also identified as a major protein band associated with miR-223 though the amount of Ago2 associated with miR-223 was slightly less than that associated with miR-16. We then analyzed the association of various miRNAs with the Ago2 complexes in the MVs by immunoprecipitating Ago2 using an anti-Ago2 antibody, followed by the detection of the miRNAs using TaqMan probe-based qRT-PCR. Interestingly, we found that the association of the MV-encapsulated miRNAs with the Ago2 complexes was variable, and among the eight miRNAs that we tested, miR-16 showed the highest percentage of total miRNA to associate with the Ago2 complexes. Combining this observation with the result from Figure 1D, we speculate that the degree of association of miRNAs with the Ago2 complexes is positively correlated with the resistance of the miRNAs to degradation by RNaseA. To further analyze the protection provided by Ago2 complexes to miRNAs in the MVs, we compared the stability of mature miR16 that was associated with Ago2 complexes with that of free, synthetic, mature miR-16. In this experiment, the Ago2-associated miRNAs in the MVs, including miR-16, were harvested byimmunoprecipitating the lysate of MV fraction using an anti-Ago2 antibody, and the amount of Ago2-associated miR-16 in the precipitated product was quantitatively analyzed by qRT-PCR, referring to the standard curve of miR-16. An equal amount of Ago2-associated miR-16 and free, synthetic miR-16 were then treated with RNaseA at various concentrations and for various time periods. As shown in Figure 3A.
