Western blot analysis
Thirty-five (35) dyskinetic rats were saved for this experiment, but one rat was sacrificed for reaching humane endpoints. Twenty-seven (27) rats were allotted in 4 groups and treated with L-Dopa alone (6 mg kg-1 + benserazide 15 mg kg-1, s.c., n=6), or L-Dopa combined with CCG-203920 (10 mg kg-1, i.p., n=7), AT-403 (0.03 mg kg-1, n=7) or CCG-203920 + AT-403 (n=7). In combination studies, CCG-203920 was administered first, followed 5 min later by AT-403 and 10 min later by L-Dopa. Western blot analysis was carried out as previously described (Arcuri et al. , 2018; Paolone et al. , 2015). Thirty minutes after L-Dopa, rats were anaesthetized with isoflurane, sacrificed by decapitation and striata rapidly dissected and frozen in liquid nitrogen and stored at -80°C until analysis. In the study where RGS4 levels were analysed, seven additional dyskinetic rats (receiving last challenge of L-Dopa 24-48 hours earlier) were also included (L-Dopa OFF group). Tissues were homogenized in lysis buffer (SDS buffer, protease inhibitor cocktail and phosphatase inhibitor cocktail) and centrifuged at 18000×g at 4°C for 15 min. Supernatants were collected, and protein levels were quantified using the bicinchoninic acid protein assay kit (ThermoScientific). Thirty micrograms of protein per sample were separated on a 4-12% gradient polyacrylamide precast gels (Bolt® 4-12% Bis-TrisPlus Gels, Life Technologies) in a Bolt® Mini Gel Tank apparatus (Life Technologies). Proteins were then transferred onto polyvinyldifluoride membrane, blocked for 60 min with 5% non-fat dry milk in 0.1% Tween20 Tris-buffered saline and incubated overnight at 4°C with anti-Thr202/Tyr204-phosphorylated ERK1/2 (pERK) rabbit monoclonal antibody (Merck Millipore, cat. #05-797, 1:1000), anti-ERK1/2 (totERK) rabbit polyclonal antibody (Merck Millipore cat. #06-182, 1:5000), anti-phospho-Ser845 GluR1 (pGluR1) rabbit polyclonal antibody (PhospoSolution, #p1160-645 1:1000), antiGlutamate receptor 1 (totGluR1) rabbit polyclonal antibody (Merck Millipore #AB1504, 1:1000), anti-Tyrosine hydroxylase (TH) rabbit monoclonal antibody (Merck Millipore, #AB152,1:1000), anti-αtubulin (αTub) rabbit monoclonal antibody (Merck Millipore, #041117, 1:25000). Membranes were washed, then incubated 1 h at room temperature with horseradish peroxidase-linked secondary antibodies (Merck Millipore, cat. #12-348, 1:2000). Immunoreactivity was visualized by enhanced chemiluminescence detection kit (Perkin Elmer), and images were acquired using the ChemiDoc MP System quantified using the Image Lab Software (Bio-Rad). Membranes were then stripped and re-probed with rabbit monoclonal anti-tubulin antibody (Merck Millipore, cat. #04-1117,1:50000). Data were analyzed by densitometry, and the optical density of specific total ERK, total GluR1, RGS4 or TH bands was normalized to the corresponding tubulin levels. Optical density of specific pERK and pGluR1 bands were normalized on totERK and totGluR1 levels, respectively (Arcuri et al. , 2018; Paolone et al. , 2015).