Protein extraction and Western blot analyses
Bilateral punches of cNAc and sNAc of 63 animals were sonicated using a cold buffer containing 0.32 M sucrose, 1 mM Hepes solution, 0.1 mM EGTA, 0.1 mM PMSF, pH = 7.4, in presence of a complete set of protease inhibitors and a phosphatase inhibitor cocktail. Total proteins have been measured in the whole homogenate by the Bio-Rad Protein Assay (Bio-Rad Laboratories). Western blots were run as previously described (Caffino et al. 2016). Briefly, ten micro-grams of proteins for each sample were run on a sodium dodecyl sulfate-8% polyacrylamide gel under reducing conditions and then electrophoretically transferred onto nitrocellulose membranes (GE Healthcare, Milan, Italy). Blots were blocked 1 h at room temperature with I-Block solution (Life Technologies Italia, Italy) in TBS + 0.1% Tween-20 buffer and then incubated with antibodies against the total proteins of interest.
The conditions of the primary antibodies were the following:
Anti vGlut1 (1:1000, Cell Signaling Technology Inc., RRID: AB_2797887), anti GLT1 (1:5000, AbCam, RRID: AB_1566262), anti GluN1 (1:1000, Invitrogen, RRID: AB_2533060), anti GluN2B (1:1000, Santa Cruz Biotechonology, RRID: AB_670229), anti GluN2A (1:1000, Invitrogen, RRID: AB_2536209), anti SAP102 (1:1000, Cell Signaling Technology Inc.), anti GluA1 (1:2000, Cell Signaling Technology Inc, RRID: AB_641040), anti GluA2 (1:2000, Cell Signaling Technology Inc., RRID: AB_10622024), anti SAP97 (1:1000, AbCam, RRID: AB_2091910), anti GRIP (1:2000, Synaptic system, RRID: AB_887728) and anti β-Actin (1:10000, Sigma-Aldrich, RRID: AB_476697).
Results were standardized using β-actin as the control protein, which was detected by evaluating the band density at 43kDa. Immunocomplexes were visualized by chemiluminescence using the Chemidoc MP Imaging System (Bio-Rad Laboratories). Gels were run 2 times each and the results represent the average from 2 different runs.