Figure 6. The main portion of serum extracellular proteasome is not stored in isolated EVs.

(a) Representative Western blot analysis (n = 3) of p2p3 (large vesicles), p4 (small vesicles) and p2p4 (all vesicles) protein extracts are shown. They derive from healthy donor’s freshly-isolated plasma and serum samples. Cell lysate from lymphoblastoid cell lines is used as control. (b) The quantitative evaluation of extracellular proteasome concentration is here shown and is obtained by applying ELISA on the fresh plasma (n = 5) and serum (n = 3) or frozen serum (n = 3) of healthy donors, or on their corresponding EV-free supernatants, which are obtained by removing the p2p4 vesicles upon ultra-centrifugations. (c) The correlation between the proteasome concentration in the total serum and the EV-free (p2p4-free) serum supernatant of a subset of German healthy donor (n = 11) and CIS patient (n = 14) cohorts (measured by ELISA) is shown. A statistically significant positive correlation is observed between the extracellular proteasome concentration in the EV-free (p2p4-free) serum supernatant and in the unfractioned serum (Pearson’s test; in healthy donors, p = 0.001; in CIS patients, p < 0.001). (d) The proteasome concentration in p2p4 vesicles of a subset of healthy donors (n = 10) and CIS patients (n = 11) is shown. It is measured by Western blotting and by using anti-α6 proteasome subunit antibody. It is expressed as proteasome concentration relative to that of the cell lysate of lymphoblastoid cell lines. No correlation between the extracellular proteasome concentration measured in serum p2p4 vesicles or in whole serum is observed. Pearson ’s test C values are reported in each chart.