Pa5 47879

Mouse tissues were homogenized and epididymal sperm were sonicated in RIPA buffer with protease inhibitor (A32963, Pierce) and 1 mM PMSF. Tissue protein lysates were separated by 4–20% SDS-PAGE gel and transferred to polyvinlylidenedifluoride (PVDF) membranes (Bio-Rad). The membranes were blocked in 5% non-fat milk, subsequently incubated with primary antibodies in blocking solution overnight at 4 °C. After washed in TBST, the membranes were incubated with HRP conjugated goat anti-rabbit IgG (1:5000, 1706515, Bio-Rad) or HRP conjugated rabbit anti-goat IgG (1:5000, 1721034, Bio-Rad) for 1 h. Membranes were incubated with chemiluminescent substrates (Bio-Rad) for detection in a molecular Imager ChemiDoc XRS+ imaging system (Bio-Rad) after 3 washes in TBST. β-Actin was visualized by incubating with anti-β-Actin-HRP (1:10000, A3854, Sigma-Aldrich) after stripping and washing. The primary antibodies used are as follows: rabbit anti-SYPL1 (1:2000, Abcam, ab184176), rabbit anti-VAMP2 (1:10000, ab181869, Abcam), rabbit anti-VAMP3 (1:10000, ab43080, Abcam), rabbit anti-VAMP4 (1:5000, 10738-1-AP, Proteintech), rabbit anti-AIF (1:1000, 5318, Cell Signaling Technology), rabbit anti-HK1 (1:4000, ab3543, Millipore), rabbit anti-PGK2 (1:5000, ab183031, Abcam), goat anti-PRSS21 (1:500, PA5-47879, ThermoFisher).

The sperm smear was fixed with 10% formalin for 10 min at RT. After rinsed in 0.01 M PBS, the smears were incubated with 0.5% Triton X 100-PBS for 5 min, then non-specific binding sites were blocked with 5% NGS in 0.2% Triton X 100-PBS for 30 min at RT. Subsequent steps for immunofluorescence of the smears were performed as described above. The primary antibodies used are as follows: rabbit anti-SYPL1 (1:50, Abcam, ab184176), rabbit anti-VAMP3 (1:100, ab43080, Abcam), rabbit anti-HK1 (1:100, ab3543, Millipore), rabbit anti-PGK2 (1:100, ab183031, Abcam), goat anti-PRSS21 (1:50, PA5-47879, ThermoFisher). After washing in PBST for 10 min × 3 times and in PBS for 5 min × 2 times, the sections were incubated with fluorescent secondary antibodies, and then mounted as above. Fluorescence was observed and photographed using EVOS FLc microscope (Life Technologies).