FIG. 1.

Peroxiredoxins are present in human spermatozoa and seminal plasma. A) Percoll-washed spermatozoa were solubilized in SDS-PAGE sample buffer (reducing conditions) and solubilized proteins from 0.1–0.5 × 10⁶ spermatozoa were loaded in each well (depending on the PRDX). B) Seminal plasma collected from the top of the Percoll gradient was diluted 1:25 in PBS (pH 7.4), centrifuged at 21 000 × g for 5 min, and finally supplemented with electrophoresis sample buffer (with reducer). Immunoblotting using the anti-PRDX1, anti-PRDX4, anti-PRDX5, or anti-PRDX6 antibody was performed as described in Materials and Methods. Specificity of the PRDX1 antibody was ascertained by preincubation with the antigenic peptide. The anti-mouse IgG antibody alone did not recognize any protein bands. Results of one experiment representative of five others (n = 6) using semen from different donors are shown. HeLa cells solubilized in electrophoresis sample buffer were used as positive control. The position of molecular mass standards (× 10⁻³) is given on the left of each immunoblot.