Figure 5

Immunodetection of MRP in as-isolated and oxidized forms with antibodies enriched for the corresponding oxidized MRPs. (A, B) 2 μg of MRPs (as-isolated and oxidized forms) were resolved on SDS-PAGE gels, transferred onto PVDF membranes, and analyzed by Western blot assays with the antibodies against indicated oxidized MRPs. Top panel: Polyclonal antiserum (non-enriched) used to detect oxidized MRP4 (A) or MRP6 (B). Middle panel: Polyclonal antiserum against oxidized MRPs was purified on an affinity column containing reduced MRP, and the unbound fraction (designated enriched) was found to be enriched for the antibodies specific for the oxidized MRPs. Bottom panel: protein loading. “R” indicates reduced and “O” indicates oxidized MRPs. (C) 2 μg of MRP4 were oxidized with indicated concentrations of H₂O₂ for 12 h at room temperature and detected with non-enriched (top panel) or enriched (bottom panel) antibodies for oxidized MRP4 by Western blot analysis. (D) Western blot analysis of different amounts of MRP4 oxidized with 10 mM H₂O₂ for 12 h at room temperature with non-enriched and enriched antibodies for oxidized MRP4. Triangles represent increasing amounts of MRP loading (0.25, 0.5, 1, 2 and 4 μg). “MRP4-R” indicates the as-isolated MRP4 (lanes 1 to 5), and “MRP4-O” indicates the oxidized MRP4 (lanes 6 to 10).