Fig. 4: Inhibitor-based profiling of EV-associated proteases responsible for α-syn cleavage.

A–C Human α-syn PFFs (hPFFs) were incubated with EVs at 1:2 ratio at 37 °C for 24 h and analyzed by Western blotting using the Syn-1 in A and C or C-20 antibodies in (B). D, E Mouse α-syn PFFs (mPFFs) were incubated with either membranous (M) or luminal (L) EV fractions and analyzed by Western blotting using the Syn-1 antibody. Proteolytic activities of different specificities were abolished by addition of corresponding specific inhibitors as following: 1,10 phenanthroline (phe), the broad-spectrum matrix metalloprotease inhibitor marimastat (mar), inhibitor specific for MMP2 (mmp2i), chymostatin (chy), a cysteine protease inhibitor E-64, inhibitor specific for MMP9 (mmp9i), aprotinin (apr), leupeptin (leu), epoxomicin (epox), lactacystin (lact), bestatin (best) and pepstatin A (pepstatA). F 100 ng monomeric α-syn (mono) (lane 1) were incubated at 37 °C for 21 h with 200 ng EVs (exo) (lane 2) in the presence and absence of specific protease inhibitors. The 8-hydroxy-5-nitroquinoline specific inhibitor of cathepsin B (CTSB I, lane 3) and LHVS inhibitor of cathepsin S (CTSS I, lane 4) were also added in combination (lane 5). The E-64 general inhibitor of cysteine proteases (lane 6), the calpain inhibitor I (CI, lane 7), and cathepsin D inhibitor pepstatin A (lane 8) were also tested at the concentrations mentioned above. Two different preparations of EVs were used in quadruple experiments. C20 was used for the detection of α-syn.