FIG. 2.

ManLAM activates functional NF-κB. (A) RAW 264.7γNO(−) cells were stimulated with ManLAM (10 μg/ml) for 30 min, and nuclear proteins were isolated, followed by an EMSA with a ³²P-labeled NF-κB consensus oligonucleotide. Compared to unstimulated cells, ManLAM induced NF-κB activation. The specificity of the NF-κB complex was verified by coincubation of the nuclear extract-oligonucleotide mixture with an anti-p50 antibody (α-p50). The nonspecific (NS) band was present in both unstimulated and ManLAM-stimulated cells and did not supershift with the anti-p50 antibody. (B) Unstimulated and ManLAM-stimulated RAW 264.7γNO(−) cells were stained with DAPI for nuclear detection and immunostained with an anti-p50-NF-κB antibody. The cells were then viewed under a fluorescent microscope at selective wavelengths to detect nuclear staining (i and iv), p50-NF-κB staining (ii and v), or both (iii and vi). After counting of 200 cells in random fields, the percentage of cells with positive nuclear staining for p50-NF-κB was 12.5% for unstimulated cells and 46.3% for ManLAM-stimulated cells. (C) RAW 264.7γNO(−) cells were transfected with 2 μg of the κB-SEAP plasmid. At 0 and 24 h after stimulation with) ManLAM (10 μg/ml), the supernatant was assayed for alkaline phosphatase activity (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Data shown in panels A and B are representative of three independent experiments. Data shown in panel C are means ± SD from three independent experiments.