FIG. 4.

Characterization of NF-κB activation by TNF-α or during HBV replication. HepG2 cells were either nontransfected (nontransf.) or transfected with wild-type or HBx⁻ mutant HBV genomic DNA for 3 days. Cells were treated with 5 ng of TNF-α per ml for 30 min as indicated. Nuclear extracts were prepared, and equal amounts (10 μg) were used to assay NF-κB DNA binding activity by EMSA with a ³²P-labeled double-stranded oligonucleotide probe containing a single NF-κB binding site. Unlabeled-competitor (cold comp.) ablation of NF-κB binding was performed by using a 100-fold molar excess of unlabeled NF-κB oligonucleotide. Complexes were resolved by electrophoresis and autoradiography as described previously (56). Complexes were quantified by densitometry.