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.