Analysis of TNF-induced NF-κB activation in normal and HPV16 or 18-immortalized keratinocytes. Subconfluent cultures of normal and HPV16 or 18-immortalized keratinocytes treated with 2 nM of TNF for 1 h were used to obtain nuclear protein extracts. For each EMSA reaction, 5 μg of nuclear protein were incubated with 50 fmol of [γ-32P]ATP-labeled double-stranded oligonucleotide and a 50X excess of unlabeled oligonucleotide (lanes 3 and 7). Specificity of binding was further demonstrated by incubation of 1 μg of nuclear protein with the described amount of labeled consensus oligonucleotide and a 50X excess of a labeled oligonucleotide carrying a single-base mutation at the NF-κB binding site (lane 4), and incubation of nuclear extract in the absence of any labeled probe (lane 8). NF-κB DNA binding reactions were carried out as described under "Material and Methods". DNA binding complexes are indicated.