Ab85104

The hypothalamus tissue samples were isolated and homogenized in ice-cold RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with proteinase inhibitors, and then subjected to centrifugation at 12,000 rpm for 15 min at 4°C to collect the supernatant. A BCA protein assay kit (P0012, Beyotime, Shanghai, China) was used to measure protein concentration. SDS-PAGE separated proteins were transferred onto PVDF membranes (Merck Millipore). The membranes were then blocked with tris-buffered saline (TBS) containing 0.1% Tween-20 and 5% nonfat powdered milk for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibodies for AGRP (1:500, ab113481, Abcam), POMC (1:2,000, 66358-1-Ig, Proteintech), GHSR (1:500, ab85104, Abcam), or α-tubulin (1:4,000, AC007, ABclonal). After primary incubation, appropriate horseradish peroxidase (HRP)-labeled secondary antibodies (1:10,000, ABclonal) were incubated with membranes for 1 h at room temperature. Signal revelation was performed using an enhanced chemiluminescence substrate (RM00021, ABclonal) detection system. Band intensities were quantified using the ImageJ analysis program.