NF-κB nuclear translocation in toxin-treated cells.

<p>Nuclear extracts were obtained from macrophages incubated with the <i>O</i>. cf. <i>ovata</i> toxin extract (final OSTRTX concentration 2 ng/ml and 20 ng/ml) (lanes 2–7) or PLTX (2 ng/ml) (8–10) for different lengths of time (i.e. 1 h, 2 h and 4 h). Cells exposed to the vehicle 4 h were used as control (lane 1). Nuclear extracts (5 µg) were submitted to EMSA using a ³²P-labeled ODN, containing the NF-κB consensus sequence (Igκ), as probe. Protein-DNA complexes were separated on 5% PAGE and then detected in a GS-250 Molecular Imager. Specificity of binding was assessed by competition experiments where nuclear extracts, from PLTX-treated cells, were pre-incubated with a 50-fold excess of cold Igκ probe (lane 11) or an ODN containing the consensus sequence of the unrelated factor YY1 (lane 12). For supershift assay nuclear extracts were pre-incubated with antibodies against NF-κB p65 and p50 subunits (lanes 13, 14), while an antibody against YY1 was used as control (lane 15).