Bis tris wells

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).