Azure 600 system

Total cellular proteins were lysed using RIPA buffer (Beyotime), loaded on an SDS‐PAGE gel, and transferred to PVDF membranes (Millipore). Primary antibodies against ALKBH1 (Abcam, ab126596, 1:1000), MFAP2 (Abclonal, A10230, 1:1000), BRD9 (CST, 58906 S, 1:1000), MPZL1 (CST, 9893 S, 1:1000), RIN1 (Abclonal, A13791, 1:1000), TMED9 (Abclonal, A3442, 1:1000), TRA2A (Absin, abs101809, 1:1000), CTSB (Abclonal, A19005, 1:1000), CMTM6 (Absin, abs148622, 1:1000), CLK3 (Absin, abs133041, 1:1000), and the internal control GAPDH (Diagbio, db106, 1:1000, Hangzhou, China) were used. After incubation with HRP‐linked rabbit secondary antibodies (Diagbio, db10002, 1:2500) and development with ECL substrate, the blots were visualized by chemiluminescence using the Azure 600 System (Azure Biosystems).

Equal numbers of brain-derived EVs and the corresponding brain tissue lysate (normalized to protein concentration) were solubilized with 5 × Laemmli sample buffer (10% SDS, 1 mg/mL bromophenol blue, 250 mM Tris pH 6.8, 5% β-mercaptoethanol, 0.5 M DTT, 50% glycerol), followed by heating to 95 °C for 8 min. Protein separation was performed using pre-cast gels 4%–20% (mini-PROTEAN TGX, Biorad, Hercules, CA, USA) or SDS-PAGE gels (8% or 10%) and transferred to nitrocellulose membranes. Nonspecific binding was inhibited by blocking with 5% NFDM (Blotting-grade blocker, Biorad) for 1 h. Blots were then probed with primary antibodies overnight at 4 °C and incubated with an HRP-conjugated secondary antibody. Either SuperSignal™ West Femto Substrate (ThermoFisher, Waltham, MA, USA) or Clarity Western ECL Substrate (Biorad) were used for membrane development, and images were acquired on a ChemiDoc imaging system (Biorad) or Azure 600 system (Azure Biosystems, Dublin, CA, USA). Primary antibodies used were CD9, CD63 (ExoAb Antibody Kit, Systems Biociences, Palo Alto, CA, USA) at a dilution of 1:1000, GM130 (sc-55591, Santa Cruz Biotechnology, Dallas, TX, USA) at 1:200 dilution, ASM at 1:500 dilution (14609-1-AP, Proteintech), Flotillin 1 (15571-1-AP, Proteintech, Rosemont, IL, USA), Calnexin (sc-46669, Santa Cruz) at 1:200, and Alix (sc-53540, Santa Cruz) at 1:100.