Substrate chromogenic solution

The spermatozoa samples were precipitated and lysed using cooled radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor to lyse the cells and then centrifuged at 10,000 × g and 4°C for 12 minutes to collect the protein. The protein was quantified by the bicinchoninic acid (BCA) method (Beyotime Biotechnology, Shanghai, China), and the loading buffer was added. After mixing, the cells were soaked in 100°C water for 5 minutes and cooled on ice. After sodium dodecyl sulfate-PAGE, the protein was transferred onto a polyvinylidene difluoride membrane, blocked in 5% nonfat milk for 1 hour, incubated with ABHD2 antibody (1:1000, ab230417, Abcam) overnight at 4°C, washed in PBS 3 times (10 minutes each), and incubated with horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG secondary antibody for 1 hour at room temperature. Then, enhanced chemiluminescence (ECL) with a substrate chromogenic solution (Thermo Fisher Scientific) was used to detect the signal by using the Bio-Rad Gel Doc XR+ (BioRad Laboratories, Hercules, CA, USA), and intensity was quantified using ImageJ (National Institutes for Health, Bethesda, MD, USA).

To investigate whether the mimetic peptide can mimic 6′-SL, the mimetic or scrambled peptides were coated in 96-well plates at 20 μg/mL in PBS overnight at 4°C, and thereafter blocked with Carbo-free™ Blocking Solution (Cat# SP-5040, Vector Laboratories) for 2 hours at room temperature. After washing 5 times for 5 minutes each with PBS containing 0.1% Tween-20, wells were incubated overnight at 4°C with different dilutions (1:20, 1:100, 1:200, 1:500) of a 6′-SL/biotinylated Sambucus nigra lectin stock solution (Cat# B-1305-2, Vector Laboratories). After washing 5 times with PBS containing 0.1% Tween-20, wells were incubated with 100 μL horseradish peroxidase-coupled streptavidin (1:3000, Cat# BA1088, Boster, Wuhan, China) for 1 hour at 37°C. After washing 6 times with 0.1% PBS containing 0.1% Tween-20 for 5 minutes each at room temperature, wells were incubated with 2,2′-azinobis-(3-et hylbenzthiazoline-6-sulphonate) chromogenic substrate solution Substrate Chromogenic Solution (Cat# 37615, Thermofisher Scientific, Waltham, MA, USA) containing 30% H₂O₂ for 30 minutes at room temperature. Absorbance was determined with an enzyme-linked immunosorbent assay (ELISA) reader at 490 nm (Tecan Infinite^® M1000 Pro).