Universal hood 2 electrophoresis imaging cabinet

The concentration of testicular total proteins obtained above was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Nantong, China). About 50 µg of protein from each sample (n = 3 for each group) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto immuno-blot polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membrane was then blocked in tris-buffered saline with tween 20 (TBST) buffer containing 5% milk for 2 h at room temperature and incubated overnight at 4 °C with primary antibody using the dilutions listed in Table 1. After 1 h of rewarming the next day, the blots were then incubated with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, CWBIO, Beijing, China). The bonded proteins were visualized with a chemiluminescent HRP substrate (Millipore, MA, USA) using a Universal Hood II Electrophoresis Imaging Cabinet (Bio-Rad, Milan, Italy), and quantified using Quantity One 4.62 software (Bio-Rad, Milan, Italy).

First, we determined the concentration of total protein in each sample (n = 5 for each group) using a Bicinchoninic Acid Protein Assay Kit (Beyotime, Shanghai, China). 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate 30 μg of protein from each sample. Then, the protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). Membranes were then blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibody overnight at 4°C using the dilutions listed in Table 1. After being rewarmed, the membranes were then incubated with species-matched horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 5000, CWBIO, Beijing, China) for 2 h at room temperature. Then, the protein signals were developed using the Universal Hood II Electrophoresis Imaging Cabinet (Bio-Rad, Milan, Italy). Immunoreactive bands were then quantified using Quantity One 4.62 software (Bio-Rad).