Girk2

Immunoblotting procedures and quantitative analysis of protein levels from mPFC punches (2 mm diameter, 2 mm thick) were performed as described (Hearing et al. 2013 (link)), using the following primary antibodies diluted in 5% milk/TBS/0.1% Tween-20 or 1% milk/TBS/0.1% Tween-20: GIRK1 (1:100, Alomone Labs; Jerusalem, Israel), GIRK2 (1:200, Alomone Labs), GIRK3 (Frontier Institute Co., Ltd.; Ishikari, Hokkaido; Japan), GABA_BR1 (1:500)(Kulik et al. 2003 (link)), GABA_BR2 (1:10, NeuroMab; UC Davis/NIH, CA), GABA_BR2 (pSer-783) (1:200, PhosphoSolutions; Aurora, CO), or β-actin (1:10,000; Abcam; Cambridge, MA). Donkey anti-mouse #926–32212 (1:1000–5000, LI-COR Biosciences; Lincoln, NE) or anti-rabbit #926–68072 (1:5000, LI-COR) secondary antibodies were used with the Odyssey infrared imaging system (LI-COR) and an integrated density of each band was measured using Image J software (NIH; Bethesda, MD).

The brain tissues were homogenized in RIPA buffer (pH 8.0) (50 mM Tris–HCl, 150 mM Nacl, 1 mM EDTA, 1% Tritonx-100, 1% Sodium deoxycholate, 0.1% SDS) supplemented with protease inhibitors (Roche Applied). After ultrasonic fragmentation for 9 s, which was performed 3 times each time, the protein extracts were centrifuged at 15,000 × g for 30 min at 4°C. The supernatants were quantified using an assay kit based on bicinchoninic acid (Thermo Fisher) and separated by 8–12% Tris–HCl SDS–PAGE gel in an electrophoresis system (Bio-Rad Laboratories). The membranes were transferred to polyvinylidene fluoride (PVDF) (Millipore) and then they were blocked with 5% not-fat milk for 1 h. The membranes were then incubated with the following primary antibodies: Pitx3 (1:1000, abcam), TH (1:500, Santa Cruz), OTX2 (1:1000, Sigma), Lmx1b (1:500, Sigma), DAT (1:1000, Millipore), Girk2 (1:1000, Alomone labs), and β-actin (1:5000; Sigma). The signals were visualized using the ECL Western Blotting Substrate (Thermo Fisher) and quantified by Image J.