Ac007

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.

Protein extracts were isolated from DPCs using a RIPA protein lysis buffer containing a protease inhibitor cocktail (Roche, Switzerland). Protein concentrations were determined using a BCA protein assay kit (Pierce, IL, United States), measuring the absorbance at 562 nm. Total protein (20 μg) was subjected to SDS-PAGE, and the separated proteins were transferred to a polyvinylidene fluoride membrane (Millipore, MA, United States). The membrane was blocked in 5% bovine serum albumin (BSA) for 1 h and probed with appropriate primary antibodies overnight at 4°C. Primary antibodies against the following proteins were used: AR (1:2,000; ab133273, Abcam, Cambridge, United Kingdom), neural cell adhesion molecule (NCAM; 1:2,000, ab75813, Abcam), β-catenin (1:2,000; ab32572, Abcam), Ki67 (1:2,000; ab16667, Abcam), cleaved caspase-3 (1:2,000; ab32042, Abcam), β-actin (1:5,000; ab8226, Abcam), and alpha-tubulin (1:5,000; AC007, ABclonal, MA, United States). After washing with Tris Buffered saline Tween, the blots were incubated with the corresponding secondary antibody (1:1,000; ab150077, Abcam) for 1 h at 20–25°C and photographed using an Odyssey infrared fluorescent scanning imager (Bio-Rad, CA, United States).