Proteolytic Potential of the MSC Exosome Proteome: Implications for an Exosome-Mediated Delivery of Therapeutic Proteasome
Figure 2
20S proteasome in MSC exosome. (a) Western blot analysis of MSC conditioned medium (CM) and exosome (Exo) using an antibody specific for PMSA 1–7 peptides. (b) Protein analysis of exosome fractionated on a sucrose gradient density. Exosome or exosome pretreated with lysis buffer was loaded on a sucrose density gradient prepared by layering 14 sucrose solutions of concentrations from 22.8 to 60% (w/v) in a SW60Ti centrifuge tube and then ultracentrifuged for 16.5 h at 200 000 g, 4°C, in a SW60Ti rotor. The gradients were removed from the top and the density of each fraction was calculated by weighing a fixed volume of each fraction. The fractions were analyzed by western blot analysis for CD9, PSMA1–7, CD81, CD59, and CD63 in exosome (upper panel) and pretreated exosomes (lower panel). (c) pI of 20S proteasome and CD9 in exosome. Exosome sample was separated by liquid-phase isoelectric focusing into 10 fractions from pH1–14. Each fraction was then concentrated and analyzed by western blot analysis for CD9, PSMA1–7. (d) Proteasome activity in MSC exosome was determined using a commercially available proteasome activity assay kit as described in the Materials and Methods section. Proteasome activity was measured by the rate of degradation of a fluorogenic peptide in the absence or presence of lactacystin, a proteasome inhibitor. One unit (U) enzyme activity is defined as the activity to generate 1 μmole product per minute at 37°C. Each bar represents mean ± SEM of 2 independent assays with each assay performed in triplicate. *.
