Tsg101

Exosome (10 μg) were electrophoresed via 4–15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After, blocking with 5% BSA, the PVDF membranes were incubated overnight at 4 °C with primary antibodies against ALG-2 interacting protein X (Alix), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA), Flotillin 2, tumor susceptibility gene 101 (TSG101), and CD81 (Novus Biologicals, Littleton, CO, USA). Signals were generated using an Enhanced Chemiluminescence (ECL) Plus system (Amersham Biosciences, Piscataway, NJ, USA). All images were detected using a Syngene PXi 4 digital imager (Syngene, Frederick, MD, USA). Quantification of protein bands was performed using Fujifilm Multi Gauge software, version 3.0.

For the immunoblot analyses of PC–NVs, equal protein amounts (10 µg) of purified PC–NVs and whole cells extracted using RIPA lysis buffer (Sigma-Aldrich) were separated by SDS-PAGE (12% gel) and transferred to polyvinylidene fluoride (PVDF) membranes. Each blot was blocked and incubated with antibodies to GM130 (BD Biosciences, San Jose, CA, USA; 1:1000), CD9 (Abcam, Cambridge, UK; 1:1000), CD81 (Novus Biologicals; 1:1000), or TSG101 (Novus Biologicals; 1:500).

Western blotting was used to identify the exosome marker (TSG101), nEV marker (CD171), and neuronal markers (TUJ1, NSE, and NeuN). For lysis, M-PER and Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) were added to each tEV and nEV sample isolated from serum. Sample concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Twenty micrograms of exosome lysates were loaded on Bolt 4–12% Bis-Tris Plus Gels (Invitrogen, Carlsbad, CA, USA) and transferred to polyvinylidene fluoride membranes (Invitrogen). The membranes were then blocked with TBS-T supplemented with 5% skim milk for 1.5 h at RT. Following blocking, the membranes were incubated with TSG101 (1:1000; Novus Bio, Littleton, CO, USA), CD171, β-actin (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), TUJ1, NeuN, and NSE (1:1000; Abcam, Cambridge, UK) primary antibodies overnight at 4 °C. After the membranes were washed with TBS-T, they were incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:2000) for 1 h at RT. Following the reaction, the membranes were again washed with TBS-T. The bands were developed using EzWestLumi Plus (ATTO, Tokyo, Japan) and analyzed using ImageQuant LAS 4000 (GE Healthcare, Buckinghamshire, UK). Western blot was performed in triplicate.