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. 1999 Jan;67(1):206–212. doi: 10.1128/iai.67.1.206-212.1999

FIG. 1.

FIG. 1

(A) Pretreatment with IFN-γ enhances the DNA binding activity of NF-κB in response to LPS. RAW 264.7 cells were preincubated with IFN-γ (50 U/ml) for 2 h and then stimulated with the indicated amounts of LPS for 30 min. Nuclear extracts (20 μg) were then analyzed by EMSA using a 32P-labeled oligonucleotide specific for an NF-κB site of the murine iNOS promoter. (B) Kinetics of activation of NF-κB by LPS. Cells were incubated with IFN-γ (50 U/ml) for 2 h before addition of LPS (100 ng/ml; indicated by the horizontal arrows; the values above the arrows indicate the lengths of LPS treatment in minutes). After additional incubation with LPS for the indicated times, nuclear extracts were prepared and analyzed by EMSA using a 32P-labeled oligonucleotide based on a functional κB site of the murine iNOS promoter. Only the region corresponding to NF-κB binding is shown. The arrowheads indicate specific NF-κB complexes. (C) Specific DNA binding of NF-κB. All of the extracts are from cells stimulated with IFN-γ (50 U/ml) for 2 h and with LPS (100 ng/ml) for 20 min (similar to panel B, lane 8). Extracts in lane 1 were not pretreated with an unlabeled oligonucleotide before analysis with a 32P-labeled oligonucleotide. Nuclear extracts were preincubated with 25-, 50-, and 100-fold molar excesses of unlabeled oligonucleotides. The wild-type (Wt; lanes 2 to 4) and mutant (Mut; lanes 5 to 7) oligonucleotides used are described in Materials and Methods.

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