Electromobility shift assay using unactivated, activated, and activated, D609-treated mouse and human cells
Nuclear extracts were prepared from unactivated, activated, or activated, D609-treated mouse (A) or human (B, C, D) cells. The extracts were incubated with radiolabeled probes containing different recognition sequences specific for either consensus NF-κB (A, B: 5′-AGT TGA GGG GAC TTT CCC AGG C-3′). In indicated cases, extracts were incubated with a 100-fold molar excess of unlabeled oligomers containing NF-κB or Apl or Sp2 binding motifs. Panel C shows the results of a representative experiment in which nuclear extract from control or activated A549 cells was incubated with an oligomer with the sequence 5′-CAA GCT GGG GAC ACT CCC 1X1-3′, which is the exact DNA sequence of the human type II NOS promoter NF-κB binding site (position — 123 through — 102). A mutated sequence (5′-CAA GCT GAA AGC ACT CCC TTT-3′) was used in D, the mutated bases are in bold type. All binding sites are underlined.