Western blotting was performed as described in a prior study.17 (link) Hypothalami and testicles were washed twice in ice-cold phosphate-buffered saline (PBS), and then dissociated in radio-immunoprecipitation assay (RIPA) buffer. The samples were then centrifuged. 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed with 50 μg of total protein from each sample, which was then transferred to membranes. After blocking, membranes were incubated with anti-Leptin-R antibody (AF497, diluted 1:1000; R&D Systems, Minneapolis, MN, USA), anti-kisspeptin antibody (ab19028, diluted 1:100; Abcam, Cambridge, UK), and anti-GPR54 antibody (abs136643, diluted 1:100; Absin, Shanghai, China). Membranes were then washed and incubated with secondary antibodies (diluted 1:5000; ZSGB-BIO, Beijing, China). Detection was performed by chemiluminescence using ECL solution (Thermo Fisher Scientific, Waltham, MA, USA). Each sample was tested at least in triplicate.