Sds protease inhibitor cocktail type 1

13 total mice (Ng^+/+ mice with saline: 3 mice, Ng^−/− mice with saline: 3 mice, Ng^+/+ mice with alcohol: 4 mice, and Ng^−/− mice with alcohol: 3 mice) were subjected to rapid CO₂ inhalation to induce unconsciousness, followed by decapitation and subsequent harvesting of brain for isolation of the NAc from both hemispheres under a surgical microscope. The extracted tissue was snap-frozen on dry ice and stored at −80°C until it was processed for SDS-PAGE (Bio-Rad Criterion system). Each NAc tissue sample was homogenized in an extraction buffer containing 50 mM Tris buffer (pH 7.4), 2 mM EDTA, 5 mM EGTA, 0.1% SDS protease inhibitor cocktail type I (Roche) and II (Sigma). Homogenates were then centrifuged at 500 g at 4°C and supernatants were collected. Protein concentration from each replicate supernatant was quantified using the Bradford protein assay (Bio-Rad). Replicates were loaded at 30 μg and separated via electrophoresis in a 4–12% poly-acrylamide gel, followed by sample preparation for proteomic analysis.

Both ENT1^+/+ mice and ENT1^−/− mice were subjected to rapid CO₂ inhalation to induce unconsciousness, followed by decapitation and subsequent harvesting of brain for isolation of the NAc from both hemispheres under a surgical microscope (n = 4 per treatment in each genotype). The extracted tissue was snap-frozen on dry ice and stored at −80°C until it was processed for SDS-PAGE (Bio-Rad Criterion system). The NAc from each mouse was homogenized using Pellet Pestle (Fisher) in a Cell-lytic MT mammalian tissue extraction reagent (Sigma-Aldrich) containing 50 mM Tris buffer (pH 7.4), 2 mM EDTA, 5 mM EGTA, 0.1% SDS protease inhibitor cocktail type I (Roche) and II (Sigma). Whole NAc tissue lysates were then centrifuged at 500 g at 4°C and supernatants were collected. Protein concentration from each replicate supernatant was quantified using the Bradford protein assay (Bio-Rad). Tissue samples were loaded at 30 μg proteins/lane and separated in MOPS buffer via electrophoresis in a 4–12% Bis-Tris poly-acrylamide gel (Criterion, Bio-Rad, Hercules CA, USA) at 70 V for 20 min, followed by 140 V for 80 min.