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).