Kinetics of Stx1-induced nuclear translocation of NF-κB complexes and IκB-α degradation. Differentiated THP-1 cells were incubated with 400 ng/mL of Stx1 for the indicated times. (A) Nuclear extracts were prepared and electrophoretic mobility shift assays were performed in the presence of a [³²P]-labeled double-stranded NF-κB binding oligonucleotide. Competition assays were performed by incubating nuclear extracts with a 25 molar excess of unlabeled oligonucleotide. Specificity of binding was assessed by incubating nuclear extracts with a radiolabeled oligonucleotide containing a substitution in the NF-κB binding motif. (B) Western blot of IκB-α degradation in the cytoplasm. TNF-α treatment of THP-1 cells for 30 minutes was a positive control for IκB-α degradation. (C) Mean NF-κB binding activity (cpm ± SEM) and IκB-α reactivity (densitometric units) from six and three separate experiments, respectively.