For western blots on whole flies or fly heads, approximately 30 frozen flies or 100 heads were homogenized in 1 mL of RIPA buffer (10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% nonidet P-40, 0.5% sodium deoxycholate, and 1% SDS) containing freshly added protease inhibitors (aprotinin, pepstatin, and phenylmethanesulfonyl fluoride). After homogenization, lysates incubated on ice for 1 hr, and were centrifuged at 4°C for 10 min. The supernatant was removed and treated for electrophoresis as described below. For western blots on mouse brain, punches of fresh nucleus accumbens tissue were frozen in liquid nitrogen. Tissue was thawed on ice and homogenized in 50 µL of RIPA buffer containing freshly added complete mini protease inhibitor tablet (Thermo Fisher Scientific, Asheville, NC). After clarifying lysates by centrifugation, a BCA protein assay (Thermo Fisher Scientific) was performed on samples. 4× LDS buffer (Life Technologies) containing β-mercaptoethanol was added to 20 µg of lysate. Samples were subjected to electrophoresis and western blotting using the NuPAGE® Novex® Tris-Acetate mini gels (Life Technologies) and the ECL Plus western blotting detection system
