Lk2001 lk2003

All testicular tissue proteins were extracted with radioimmunoprecipitation assay lysis buffer (P0013B; Beyotime Institute of Biotechnology, Haimen, China) then quantified using a bicinchoninic acid assay. Briefly, 40 µg/lane protein samples were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was blocked at 37°C for 2 h with 5% non-fat milk in TBST buffer and then incubated with the following primary antibodies at 4°C overnight: Bax (sc-493; 1:500; Santa Cruz Biotechnology, Inc.), cleaved-caspase-3 (sc7148; 1:1,000; Santa Cruz Biotechnology, Inc.), tumor necrosis factor (TNF)-α (ab6671; 1:500; Abcam, Cambridge, UK) and interleukin (IL)-1β (ab100768; 1:200; Abcam). Following three washes with TBST buffer, membranes were incubated with secondary goat anti-rabbit antibodies conjugated with horseradish peroxidase (LK2001/LK2003; 1:100; Sungene Biotech, Co., Ltd., Tianjin, China) at room temperature for 1 h. All specific bands were visualized using an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Optical densities were detected using ImageJ software version 1.48 (National Institute of Health, Bethesda, MD, USA).

Testis tissue proteins from both groups were extracted with RIPA lysis buffer (P0013B; Beyotime Institute of Biotechnology, Haimen, China) and quantified using a Bicinchoninic Acid assay (K763-KIT; Amresco, LLC, Solon, OH, USA). Equivalents of protein samples (40 µg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membrane was blocked with TBS/Tween-20 (TBST) buffer with 5% non-fat milk and then incubated at 4°C with the following primary antibodies: Bax (sc-493; 1:500), B-cell lymphoma (Bcl-2; sc-7382; 1:1,000), cytochrome c (cyt c; 1:2,000 dilution; cat. no. Santa Cruz Biotechnology, Inc.) and cleaved caspase-3 (sc-13156; 1:1,000; all from Santa Cruz Biotechnology, Inc.) at 4°C overnight. Following rinsing twice with TBST, the membranes were incubated with secondary goat anti-rabbit antibodies conjugated with horseradish peroxidase (LK2001/LK2003; 1:100; Sungene Biotech, Co., Ltd., Tianjin, China) at room temperature for 1 h. All specific bands were visualized using an enhanced chemiluminescence system (Pierce; Thermo Fisher Scientific Inc., Waltham, MA, USA). Optical densities were analyzed using the ImageJ software version 1.48u (National Institutes of Health, Bethesda, MD, USA).