Western Blotting of Synaptic Proteins

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).