Overexpression of c-rel restores IL-12 p40 induction in H₂O₂-treated macrophages. The RAW 264.7 macrophages were transfected with either the mock plasmid (PRC/CMV) or a c-rel-overexpressing plasmid construct using Lipofectamine 2000. Cells were incubated for 24 hours, washed, and further stimulated with LPS plus IFN-γ in the absence or presence of 250 μM H₂O₂. After 48 hours, the amounts of IL-12 p40 (mean ± SD) secreted in the culture supernatants were measured by EIA (A). The c-rel, p65, and p50 levels in the nuclear extracts were detected by Western blotting after 1 hour of stimulation with LPS plus IFN-γ (B). The equal loading of protein was confirmed by staining with Ponceau S stain (B). This experiment is representative of 3 experiments performed with similar results.