FIG. 7.

ManLAM-induced NF-κB binds to the 5′-flanking region of the IRF-1 promoter but does not induce IRF-1 expression. (A) RAW 264.7γNO(−) cells were stimulated with ManLAM for 30 min, and nuclear extracts were probed with a ³²P-labeled oligonucleotide that spans the NF-κB-binding site on the 5′-flanking region of the IRF-1 promoter. As shown, ManLAM-induced NF-κB binds to this site and was supershifted with the anti-p50 antibody (α-p50). (B) To determine if ManLAM is capable of inducing IRF-1 expression, the cells were stimulated with either 10 or 50 μg of ManLAM/ml for 2 or 18 h, followed by Western blotting for IRF-1 (lanes 8 to 11). For positive controls, the cells were stimulated with IFN-γ (10 U/ml) for 2 and 18 h (lanes 2 and 3). Since RAW 264.7 cells are quite sensitive to LPS, they were also stimulated with 1 and 100 ng of LPS/ml for 2 and 18 h (lanes 4 to 7). Whereas IFN-γ induced IRF-1 protein expression, neither ManLAM nor LPS did, even at relatively high concentrations. (C) RAW264.7γNO(−) cells stimulated with IFN-γ or ManLAM at the indicated concentrations and times were lysed with Laemmli sample buffer containing 2.5% SDS, which lyses both plasma and nuclear membranes; the lysates were then sonicated for 2 s with a probe sonicator, separated by SDS-PAGE, and immunoblotted for IRF-1. (D) Nuclear proteins were isolated from unstimulated cells and from cells stimulated with IFN-γ, LPS, or ManLAM at the indicated concentrations and times. The nuclear proteins were then separated by SDS-PAGE and immunoblotted for IRF-1.