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. Author manuscript; available in PMC: 2015 Jan 28.
Published in final edited form as: ACS Nano. 2014 Jan 9;8(1):483–494. doi: 10.1021/nn404945r

Figure 3. EV-GlucB exhibited EV-specific Gluc activity, and was stable in blood and urine ex vivo over time.

Figure 3

(a, b) Gluc activity assay of EV-GlucB (a) and EV-Gluc (b) vesicles following sucrose gradient fractionation. Note distinct increase in Gluc activity of EV-GlucB in EV-containing fractions (#3, 4, 5) when compared to EV-Gluc. (c, d) Western blot analysis of proteins extracted from pelleted fractions demonstrated significant Gluc expression and biotinylation of EV-GlucB (c), but not on EV-Gluc (d). Exosomal marker, Alix (95 kDa), was immunoprobed to identify exosome-containing fractions. Cell lysates of HEK293T cells stably expressing sshBirA with GlucB and Gluc were used as positive controls. (e, f) Gluc activity of EV-GlucB was stable in biofluids ex vivo over 24 h. Blood (e) and urine (f) collected from untreated animals were spiked with EV-GlucB vesicles, incubated at 37°C, and samples collected at different time points over 24 h. No significant loss of EV-GlucB signal was detected in either type of biofluid. P > 0.05 by one-way analysis of variance (ANOVA) at all time points.