FIG. 4.

Effect of A238L on the binding of p50/p65 and p300 to NF-κB sites in the iNOS promoter. (A) Nuclear extracts from Raw-pcDNA and Raw-A238L cells treated (+) or not (−) with 1 μg/ml of LPS plus 200 U/ml of IFN-γ for 6 h were incubated with the indicated biotinylated probe, and the complex was pulled down with streptavidin-agarose beads, as described in Materials and Methods. After extensive washing, proteins in the complex were analyzed by Western blotting using antibodies against p300, p65, or p50. The control probe is a biotinylated 22-bp nonrelevant DNA sequence of the murine iNOS promoter. Inputs were also included to show the presence of the analyzed proteins in nuclear protein extracts of Raw cells. (B) Raw-pcDNA (open bars) or Raw-A238L (shaded bars) cells were transiently transfected with the p(iNOS)m-luc reporter plasmid and with the indicated doses (from 0 to 1.6 μg of DNA/10⁶ cells) of two different expression plasmids: pRC-CMV-cRel to overexpress the p50 subunit of NF-κB or pCMV-p65 to overexpress the p65 subunit of NF-κB transcription factor. Sixteen hours after transfection, the cells were cultured in the absence (plain bars) or presence of 1 μg/ml of LPS plus 200 U/ml of IFN-γ (striped bars) for 6 h and assayed for luciferase activity. Extracts were normalized to Renilla luciferase activity as described in Materials and Methods. Results from triplicate assays are shown in RLU per μg of protein (means ± SD).