Azure cseries imager

To ensure the purity of the isolated SVs, 2 µL of each of the 30 separated fractions and the pellet from the SV isolation were blotted onto a nitrocellulose membrane divided into grids. Samples were added slowly, so the area of the absorbed drop of the sample was 2–4 mm in diameter. The membrane was allowed to dry before blocking non-specific sites using 5% BSA in TBS-T for 30 min to 1 h. The membrane was incubated with primary antibody diluted in 0.1% BSA in TBS-T for 30 min at room temperature. Anti-PSMC6 (1:1000; Abcam, Cambridge, UK), a proteasome marker, was used to determine fraction purity and ensure no fractions with proteasome contaminants continued into the sucrose cushion step of the isolation protocol. After incubation, the membranes were washed three times with TBS-T for 5 min each. The secondary antibody was diluted according to the manufacturer’s protocol in 0.1% BSA in TBS-T and added to the membrane for 30 min at room temperature. Secondary antibodies were HRP-conjugated anti-rabbit IgG (Thermo Scientific). The membrane was washed three times with TBS-T for 5 min each. Membranes were allowed to sit in SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) for 1 min before imaging with the Azure cSeries Imager (Azure Biosystems, Dublin, CA, USA).

Western blotting was performed as described in our previous studies [51 (link),56 (link),59 (link)]. Briefly, purified synaptosomes (7.5 µg) from each animal from the two groups were loaded onto 10% Bis-Tris wells (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen, Waltham, MA, USA) and immunodetection. Nonspecific antibody blocking was performed using Superblock (ThermoFisher, Waltham, MA, USA). Immunoblotting was performed with primary antibodies overnight at 4 °C against ADD1 (1:1000, ProteinTech, Rosemont, IL, USA) and GAPDH (1:2500, Invitrogen, Waltham, MA, USA), followed by secondary antibody (1:2500, HRP-conjugated anti-rabbit IgG; Thermo Scientific, Waltham, MA, USA) and (1:2500, HRP-conjugated anti-mouse IgG; Thermo Scientific, Waltham, MA, USA) against ADD1 and GAPDH, respectively. Primary and secondary antibody dilutions were carried out according to the manufacturer’s suggestions. Blots were developed using Azure CSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

SVs (10–20 µg) from each animal were loaded into 4–12% Bis-Tris wells (Invitrogen, Waltham, MA, USA) under reducing conditions, followed by transfer to a nitrocellulose membrane using iBlot2 (Invitrogen) and immunodetection. Nonfat milk (5%) was used to block nonspecific antibody binding (Thermo Fisher Scientific, Waltham, MA, USA). After blocking, membranes were incubated overnight at 4 °C with a primary antibody. Primary antibodies included GAPDH (Invitrogen), PSD95 (Invitrogen), GFAP (Sigma-Aldrich, St. Louis, MO, USA), SYP (ThermoFisher), VGLUT1 (Santa Cruz Biotech, Dallas, TX, USA), and SNAP25 (Synaptic Systems, Goettingen, Germany). MEGF8 (Bioworld Technology, St. Louis Park, MN, USA) and LAMTOR4 (Cell Signaling, Danvers, MA, USA) were additionally selected from the proteomic analysis for post-validation. Secondary antibodies were HRP-conjugated anti-rabbit IgG (Thermo Scientific) and HRP-conjugated anti-mouse IgG (Thermo Scientific). Primary and secondary antibody dilutions were done according to the manufacturer’s suggestion and are shown in Supplementary Table S1. Blots were developed using Azure cSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).