Supersignal west femto chemiluminescent substrate detection system

The protein was extracted using RIPA Lysis Buffer (Solarbio, Shanghai, China) and the concentration was determined using the BCA protein assay kit (Solarbio, Shanghai, China) according to the instructions. Proteins were separated using SDS-PAGE (8–12% gel). The membrane was blocked with 5% non-fat milk and incubated with primary antibodies at 4 °C overnight. The membranes were incubated with appropriate secondary antibodies (bioss, Beijing, China) at room temperature for 1–2 h. The proteins were visualized by an enhanced chemiluminescence (ECL) system (Millipore, USA) and the SuperSignal West Femto Chemiluminescent Substrate Detection System (Thermo Fischer Scientific, Rockford, IL). The relative protein levels were measured using Image J software, and normalized to Tubulin. Primary antibodies were purchased from Abcam (Abcam, Cambridge, USA) including Bax (ab32503), Bcl-2 (ab182858), Caspase-3 (ab184787), MMP-9 (ab76003), and Tublin were purchased from Elabscience (Elabscience, Wuhan, China).

Testicular lysates and immunoprecipitated proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane as previously reported (31 (link),32 (link)). The membrane was blocked and incubated overnight at 4 °C with the indicated primary antibodies (Table S1). The membrane was washed and incubated at room temperature for 2 h with secondary antibodies. The signals of the detected proteins were visualized on the SuperSignal West Femto Chemiluminescent Substrate detection system (Thermo Scientific).