P0690

The protein lysis buffer (Beyotime, P0013B, China) containing protease inhibitors (Roche, 11,697,498,001, Switzerland) was added to lysate the spermatogenic cells, and incubated on ice for 30 min. The lytic mix liquids were centrifugated at 12,000 × g for 15 min to collect the supernatants. The protein was quantified with BCA method (Beyotime, P0012S, China), then mixed with 5 × loading buffer (Beyotime, P0015, China) and denatured at 95℃ for about 10 min. Subsequently, 15 μg of total protein and 5 μL of protein Marker (P0068, Beyotime) was added to 10% SDS-PAGE electrophoresis gel (Beyotime, P0690, China), respectively. Electrophoresis was performed at 90 V and the voltage was adjusted to 120 V when the Marker ran past the concentrated gel, and then the protein was transferred to PVDF membrane. After blocked with 5% nonfat-dried milk buffer, primary antibody (PLZF/GFRA1/β-actin/SYCP3/DDX4/c-Kit) was added to change the blocking buffer and incubated overnight at 4℃. HRP-labeled secondary antibody was incubated with the membrane at room temperature in an hour, and then the ECL Chemiluminescent substrate (4A BIOTECH, w011-20, China) was added to cover the PVDF membrane. Finally, the image was recorded with chemiluminescence imager (ChemiDoc Touch, Bio-Rad).

Protein expressions of caspase-1, Capase-4, and cleaved N-terminal gasdermin D (GSDMD-N) were measured by Western blotting [26 (link)]. Proteins were extracted from cells and tissues by RIPA (Sigma-Aldrich, Missouri, USA) reagents and were detected using the BCA method (P0012; Beyotime, Shanghai, China). Next, 30 μg total protein from each well was separated by 10% SDS-PAGE electrophoresis (P0690; Beyotime, Shanghai, China) and transferred to a PVDF membrane (FFP32; Beyotime, Shanghai, China). The membranes were blocked with 5% defatted milk for 1 h. Then, primary antibodies, including anti-caspase-1 (ab207802, 1/1000), anti-Capase-4 (ab238124, 1/1000), and anti-GSDMD-N (ab215203, 1/1000), were added (Abcam, California, USA). The next day, HRP labeled secondary antibody (ab7090, Abcam, California, USA) was added after the membrane was washed with PBS, and the membrane was incubated at room temperature for 1 h. The membranes were developed using ECL reagents with GAPDH as control.

Proteins were isolated using a protein extraction kit (PROTTOT-1KT, Sigma-Aldrich) and quantitated using Bradford Protein Assay Kit (P0012, Beyotime). The proteins (30 ug each lane) were separated using SDS-PAGE gels (P0690, Beyotime) and transferred to PVDF membranes (IPVH00010, Millipore), which were blocked with TBST solution containing 3% BSA (36104ES, Yeasen, China) for 2 h. The membranes were then incubated at 4 °C overnight with appropriate primary antibodies, which were diluted by TBST solution (3% BSA). An HRP-conjugated secondary antibody and enhanced chemiluminescence luminescent solution (P0018S, Beyotime) were used to detect the protein bands. The bands gray value was measured by ImageJ. The primary and secondary antibodies used in this study are listed in Supplementary Table 2.