Involvement of CaM in H₂O₂-mediated down-regulation of c-rel. The RAW 264.7 macrophages were stimulated with LPS plus IFN-γ in the absence or presence of 250 μM H₂O₂. After 1 hour of stimulation, the cells were harvested and both the cytoplasmic and the nuclear extracts were prepared. CaM contents (mean ± SD) in the cytoplasmic extracts were determined by EIA as described in “Materials and methods” and expressed as the fold changes over unstimulated control (A). The same cytoplasmic extracts were incubated with anti-CaM antibody for 3 hours at 4°C and then protein A/G-Sepharose was added to the mixture and was further incubated for 2 hours at 4°C. Coimmunoprecipitated c-rel was detected by Western blot (B, top lane). The same preparation was also used to detect CaM level using anti-CaM antibody, respectively (B, middle lane). The equal loading of protein was confirmed by staining with Ponceau S stain (B, bottom lane). The c-rel levels in the nuclear extracts were measured by immunoblot analysis (C, top lane) of identical amounts of total proteins loaded in the blot (C, bottom lane). This experiment is representative of 3 experiments performed with similar results. In another experiment, the RAW 264.7 macrophages were either treated with LPS plus IFN-γ alone or cotreated with H₂O₂ and LPS plus IFN-γ in the absence or presence of 5 μM TFP, which is a known inhibitor of CaM activity. The nuclear accumulation of c-rel was detected after 1 hour by immunoblot analysis (D, top lane). The equal loading of protein was confirmed by Ponceau S staining (D, bottom lane). The IL-12 p40 levels (mean ± SD) secreted in the culture supernatants were determined after 48 hours by EIA (E). Results shown are representative of 3 independent experiments.