Western Blot Analysis of OB and OBR Proteins in Ram Reproductive Tissues

Western blot was used for analysis of OB and OBR proteins in testis, epididymal tissue, ejaculated and epididymal sperm of rams as described by the method of Zhang (Zhang et al., 2017 (link)). Briefly, tissues or cells were lysed with a lysis buffer (HX1862; Huaxingbo, Hong Kong, China). Equal amounts of protein were resolved using 12% SDS-PAGE gel and transferred to PVDF membranes (ISEQ00010; Millipore, Burlington, MA, USA). After they were blocked with 5% nonfat milk or BSA (A3311; Sigma-Aldrich, St. Louis, MI, USA) at 37 °C for 60 min, the membranes were incubated with primary antibodies against OB (1:1,000, ab3583, Abcam, Cambridge, UK), OBR (1:1,000, 20966-1-AP, Proteintech, Rosemont, IL, USA) and GAPDH ( 1:2,500, WL01114; Wanleibio, Hebei, China) at 4 °C, overnight. The membranes were then washed 3–5 times with TBST buffer (HX1893; Huaxingbo, Hong Kong, China) and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5,000, 7074; Cell Signaling Technology, Danvers, MA, USA). Western blotting detection reagents (GE Healthcare, Uppsala, Sweden), and then were visualized using a cooled CCD camera (Nikon, Tokyo, Japan) based chemiluminescence detector (GE Healthcare, Uppsala, Sweden). Protein bands were then analyzed by computer software (Image-Quant 350; GE Healthcare, Uppsala, Sweden).