Ab85367

Salts and organic solvents used in solution preparations were purchased from Fisher Scientific (Leicestershire, UK), Sigma Chemicals (St. Louis, MO, USA, EUA), or Merck (Darmstad, Germany), with the highest grade of purity commercially available. It used antibodies to analyze D1R, D2R, DARPP32 (ab81296, ab85367, and ab40801 respectively, Abcam, UK), and tyrosine hydroxylase (TH), (T1299, Sigma Aldrich, St. Louis, MO, USA) at a 1:1000 dilution. Moreover, antibodies against GLP-1 and GLP-1R and against the phosphorylated form of insulin receptor were used (ab22625, ab218532, and InsR-Tyr972, ab5678 respectively, Abcam, UK) as well, against phosphorylated AMPK form (Thr172, 2535S, Cell Signalling Technology, Danvers, MA, USA, EUA). Calnexin was used as loading control (AB0037, Sicgen, Cantanhede, Portugal). Plasma dopamine levels and plasma GLP-1 levels were assessed through the Dopamine ELISA Kit, (Abnova, Taiwan) and Rat GLP1/Glucagon-like Peptide 1 ELISA Kit, (LifeSpan BioScience, Inc., Washington, DC, USA) respectively.

The proteins were extracted from the striatum tissues using radioimmunoprecipitation assay (RIPA) lysis buffer (Ukzybiotech, Beijing, China) according to the manufacturer's instructions. The protein concentration in the lysates was evaluated using a BCA Protein Assay Kit. Proteins were then separated on an SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% BSA-TBST and then incubated with DA1R antibody (ab20066, 1 : 1000), DA2R antibody (ab85367, 1 : 1000), DA3R antibody (ab42114, 1 : 1000), DA4R antibody (ab20424, 1 : 500), DA5R antibody (ab40656, 1 : 200), PKA antibody (ab211265, 1 : 1000), CAM (ab45689, 1 : 1000), and CAMKII (ab52476, 1 : 1000) (Abcam) overnight at 4°C. Then, the blots were washed, incubated with 5% BSA-TBST, and washed again. Consequently, samples were incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 2 h. Band detection was performed using the enhanced chemiluminescence (ECL) detection kit. The intensity of the detected bands was calculated densitometrically using the Gel Image system ver.4.00 (Tanon, China).