Evaluation of protein levels for IZUMO1 analyzed by western blotting. (A) Western blotting analysis with 2.2 μg/mL of monoclonal anti-IZUMO1 antibody (Mab18). Sperm lysates (30 μg) were applied onto each lane from an individual derived from five mouse lines (WT/WT; a–c, WT/v1KO; d–f, v1KO/v1KO; g–i, v1KO/KO; j–l and KO/KO; m). The alphabetical letters indicate individual specimens. BASIGIN is used as an internal control. The asterisk indicates non-specific bands. (B) Relative quantification for IZUMO1 protein normalized using BASIGIN. The color scheme is as follows: WT/WT; red, WT/v1KO; orange, v1KO/v1KO; light green, v1KO/KO; blue. Significant differences: ** p < 0.01 and *** p < 0.001 (Student’s t-test) compared to WT/WT.