EMSA of NF-κB binding activity. The DNA binding activity of nuclear extracts from HepG2 cells cultured in the presence or absence of IL-1β and/or IL-4 for 30 minutes were examined using a ³²P-labeled oligonucleotide from the sIL-1Ra promoter containing the NF-κB binding site (A). Competition studies were performed with nuclear extracts from HepG2 cells stimulated with IL-1β (see Materials and Methods). Lane 1, unstimulated; lane 2, IL-1β; lane 3, IL-4; lane 4, IL-1β and IL-4; lane 5, competition with the cold probe; lane 6, competition with the cold probe mutated in the NF-κB binding site; lane 7, competition with the cold oligonucleotide containing the NF-κB consensus region. Characterization of the protein present in the NF-κB–DNA complex was performed using specific antibodies against p65(RelA) and p50 (B). Lane 1, unstimulated; lane 2, IL-1β; lane 3, preincubation of nuclear extracts of IL-1β–stimulated HepG2 cells with antibodies against NF-κB p65 (RelA) before the addition of the ³²P-labeled NF-κB probe; lane 4, preincubation with antibodies against p50; lane 5, preincubation with control IgG; lanes 6 to 8 contain the NF-κB probe and the antibodies against the p65 subunit, p50 subunit, and control IgG, without nuclear extracts. The dark arrow shows the NF-κB–DNA complex. The open arrow represents the supershifted complex. NS = nonspecific protein-DNA binding. These results are representative of three different experiments.