Fig. 4

Identification of PRDX5 and PRDX6 complexes with TLR4 in sperm cells. Anti-TLR4 monoclonal antibodies were immobilized on a HiTrap protein A HP column (Cytiva) to capture TLR4-PRDX complexes from sperm lysates. The chromatogram highlights the fraction containing protein complexes associated with immobilized TLR4 (A). After elution and concentration, the immunoprecipitated proteins were analyzed by non-reducing SDS-PAGE, followed by Western blotting. Four distinct reactive bands corresponding to PRDX5 were detected above 150 kDa (B), while 12 reactive bands corresponding to PRDX6 were observed, ranging from 17 kDa to above 250 kDa (C). The reactive bands are indicated by red arrows. These results confirm the association of PRDX5 and PRDX6 with TLR4, indicating the formation of TLR4-PRDX complexes in sperm cells