Hs201

The proteins (20 μg) of adult boar reproductive tissues and sperm were separated by SDS-PAGE using 12% (v/v) gels and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies against CRISP2 (1:1,000; SAB2501635, Sigma, USA) or β-actin (1:1,000; HC201, TransGen Biotech, China) overnight at 4°C. The membranes were washed 3 times for 10 min each with TBST (0.1% Tween 20, 20 mM Tris/HCl, 150 mM NaCl; pH 8.0) and incubated for 1 h with horseradish peroxidase (HRP)-conjugated rabbit anti-goat (1:3,000; E030130-02; Earthox, San Francisco, USA) or goat-anti-mouse (1:3,000; HS201, TransGen Biotech, China) secondary antibodies at room temperature for 1 h. The membranes were incubated for 5 min with the enhanced chemiluminescence (ECL) detection reagent in the dark and then exposed with a Tanon-5200 Imaging System (Tanon, Shanghai, China). β-actin was used as the internal control, and the relative protein expression levels of CRISP2 were analyzed by using ImageJ software (https://imagej.nih.gov/ij/index.html).

To quantify protein expression by western blot, a polyhistidine-tag (6 × His-tag) was attached to C terminals of each protein. Strains were cultured in SC–Ura medium with the appropriate carbon source at 30 °C for 20 h. Then 10 A₆₀₀ units of yeast cell were harvested, washed by water and resuspended by 500 μl of 0.3 M NaOH. After incubation at room temperature for 5 min, cells were washed with 500 μl water, spun down and resuspended in 100 μl lysis buffer (4% β-mercaptoethanol, 0.06 M Tris–HCl, pH 6.8, 2% SDS, 5% glycerol), boiled for 10 min and centrifuged again at highest speed (14,000×g). After protein quantification by Bradford method, 10 μl supernatant (containing ~40 μg total protein) was loaded per lane of 12% SDS–PAGE gel. Proteins were transferred to a PVDF membrane and blocked by 5% BSA buffer. Anti-His antibody (1:2000, TransGen HT501, China) and anti-GAPDH antibody (1:2000, TransGen HC301, China) were used to probe target proteins and internal reference protein GADPH. Goat anti-mouse IgG (H + L) HRP-conjugated antibody (1:3000, TransGen HS201, China) was used as the secondary antibody. Multi-Chemiluminescent Blot Imaging System (Tanon-4800, China) was used for image collection. ImageJ software was used to quantify the intensities of protein bands (https://imagej.nih.gov/ij/).