2.5. Western blot analysis of dopamine D2 and D4 receptor, glucose transporter 1 and 3, and melanocortin 4 receptor

Hypothalamic tissue (0.1 g) was washed three times with KCl buffer (1.15%) and then homogenized in 450 µl 1.15% KCl, gently shaken on ice for 30 min and centrifuged at 13,000 g for 10 min at 4°C. The cytosolic supernatant was stored at −80°C for the determination of glucose, albumin, lactate, pyruvate, and the malondialdehyde test. The pellet was suspended in 200 µl RIPA buffer with 4% Triton‐X100 (Sigma‐Aldrich), 1 mM PMSF (Sigma‐Aldrich), and Complete Protease Inhibitor Cocktail Tablets (Roche Diagnostics) overnight at 4°C with gentle shaking (Wang et al., 2010). The lysate was centrifuged at 13,000 g for 10 min at 4°C. Total protein content of the supernatant was measured using the DC Protein Assay from Bio Rad. Twenty‐five microgram protein from each animal was separated on NuPAGE™ 4%–12% Bis‐Tris gels (Thermo Fisher scientific) and blotted onto Invitrolon membranes (Thermo Fisher scientific). Membranes were then blocked with Roti Block (Carl Roth) in TBS‐T (1:10) at 4°C for 4 hr. The blocked PVDF membranes were incubated overnight at 4°C either with rabbit polyclonal D2R antibody (1:1,000; Santa Cruz Biotechnology, RRID: sc‐9113) or rabbit polyclonal D4R antibody (1:1,000; Abcam, RRID: ab20424) antibody in TBS‐T and then probed at room temperature for 1 hr with the HRP‐conjugated secondary antibody (1:10,000; Abcam; RRID: ab97051, m). Resulting autoradiographs were quantified by densitometry using Gene Snap and Gene Tools software (Syngene, Synoptics Ltd). The membranes were routinely stained with Ponceau S before immunostaining, in order to confirm correct samples loading and the transfer of equivalent amounts of protein (Romero‐Calvo et al., 2010).

Expression of MC4R, Glut1, and Glut3 was determined on the same membranes after stripping with Roti‐Free stripping buffer 2.0 (Carl Roth). The overnight incubation was done with mouse monoclonal MC4‐R antibody (1:400; Santa Cruz Biotechnology, RRID: sc‐55567), rabbit polyclonal Glut1 antibody (1:500; Santa Cruz Biotechnology, RRID: sc‐7903), and Glut3 antibody (1:500, Santa Cruz Biotechnology, RRID: sc‐30107).