Universal protein extraction lysis buffer

Cells were lysed with universal protein extraction lysis buffer (Bioteke, Peking, China) which contained protease inhibitors (Roche, Basel, Switzerland). Protein lysates were run on 8–12% SDS PAGE gels and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was incubated at room temperature for 1 h in 3% BSA and then blotted with specific primary antibody overnight at 4 °C. After incubation for 1 h at room temperature in the HRP-labeled secondary antibody, the membrane was detected with chemiluminescence Western blot detection system (Millipore, Billerica, MA, USA).

Proteins of the mouse testicular tissues were extracted using a universal protein extraction lysis buffer (Bioteke, Winooski, USA) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). The denatured proteins were separated on 10% SDS-polyacrylamide gels and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, USA). Then, the membrane was blocked for 0.5 h at room temperature and incubated overnight at 4°C with the following primary antibodies: anti-Cep112 (1:1000; ThermoFisher), anti-Na/K-ATPase (1:500; Abclonal), anti-Spata6 (1:1000; ThermoFisher), anti-Wdr60 (1:500; ThermoFisher), anti-Akap3 (1:1000; Abcam), and anti-Tubulin (1:500; Abclonal) antibodies. After being washed with 0.3% TBST, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibodies (1:5000; Abclonal) for 1 h at room temperature. Then the blots were visualized with the gel imaging system (Beijing Liuyi Biotechnology, Beijing, China).