H₂O₂ increases nuclear p50 and p65 NF-κB expression but inhibits c-rel levels in the nucleus. The RAW 264.7 macrophages were treated with 250 μM H₂O₂ for 1 hour followed by stimulation with LPS plus IFN-γ for another 1 hour. Nuclear extracts were prepared and the expression of p65 (A, top lane) and p50 (A, middle lane) component of NF-κB was examined by immunoblot analysis. As a loading control identical amounts of total proteins were loaded (A, bottom lane). The same nuclear extracts were used to examine c-rel expression by Western blotting (B). This experiment is representative of 5 experiments performed with similar results.