Assays

The exosome pellets were lysed in 80 µL of a modified RIPA lysis buffer (50 mM TrisHCl pH 8, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v)) with protease inhibitors (Sigma Aldrich, US) on ice with intermediate vortexing. 20 µL H2O, 10 µL 100 mM MgCl2, and 25 units Benzonase (Sigma Aldrich, US) were added followed by an incubation for 30 min at 37°C at 1400 rpm in a Thermomixer (Eppendorf, Germany). Undissolved material was removed by centrifugation for ...

The neurons were lysed direct on the plate in RIPA lysis buffer using a cell scraper. The lysate was transferred into a fresh Eppendorf tube and undissolved material was removed by centrifugation for 5 min at 20,000 g and 4°C. A protein assay was performed and 15 µg of protein were subjected to proteolytic digestion with the SP3 protocol. Proteins were reduced by addition of 9 µL of 200 mM dithiothreitol (Biozol, Germany) in 50 mM ammonium bicarbonate and incubation for 30 min at 37°C. Cysteine ...