FIG. 5.

dsRNA- and TNF-α-induced specific DNA-binding activity of NFκB. pkr^+/+(EX12) MEF were treated with Lipofectin alone (lanes Co), with pI-pC (10 μg/ml) in the presence of Lipofectin (lanes dsRNA), or with TNF-α (lanes TNF). At 3 h after either Lipofectin or dsRNA treatments or 20 min after the TNF-α treatment, the cells were harvested and nuclear extracts were prepared as described in Materials and Methods. EMSAs were performed as described in Materials and Methods. Where indicated, a 100-fold molar excess of either the specific NF-κB-binding nonlabeled oligonucleotide (specific competitor) or a p53-binding nonlabeled oligonucleotide (nonspecific competitor) was added to the binding-reaction mixtures for 10 min before the addition of the specific ³²P-labeled NF-κB-binding oligonucleotide. In the last lane, the undiluted anti-p65/RelA (C-20 from Santa Cruz) antibody was added in 1/10 of the final reaction volume for 10 min before the addition of the specific ³²P-labeled NF-κB-binding oligonucleotide. Addition of several irrelevant antibodies did not interfere with the specific binding of NF-κB to DNA, demonstrating the specificity of the anti-p65/RelA antibody-induced supershift (data not shown).