FIG. 3.
(A) Effect of CHX on induction of the DNA binding of NF-κB by LPS with or without pretreatment with IFN-γ. RAW 264.7 cells were primed with IFN-γ (50 U/ml) for 30 min in the presence or absence of CHX (25 μg/ml) (lanes 4 and 5). LPS (100 ng/ml) was then added, and the mixture was incubated for 20 min. Nuclear extracts were prepared and analyzed by EMSA as described in the legend to Fig. 1. (B) Effect of antibodies against TNF-α or IL-1R on the induction of DNA binding of NF-κB by LPS with or without priming by IFN-γ. An anti-TNF-α antibody (2.5 μg/ml) and/or an anti-IL-1R antibody (2.5 μg/ml) were added to the culture 30 min before priming with IFN-γ (50 U/ml) for 60 min. LPS (100 ng/ml) was then added for an additional 20 min before the preparation of nuclear extracts. Extracts were then analyzed by EMSA as described in the legend to Fig. 1. (C) Synergistic activation of NF-κB by TNF-α or IL-1 in association with IFN-γ (50 U/ml). Cells were treated with the indicated agents as described above. Cells were stimulated with recombinant IL-1 (25 ng/ml) and TNF-α (10 ng/ml) for 20 min in this experiment. In all cases, only the relevant region of the autoradiogram is shown.