Figure 3

Exposure of MDM to opsonized and unopsonized CS2 IE activates NF-κB to similar extents. MDM (1x10⁶) were primed with IFN-γ for 48 hr, treated with media alone (MDM), 2x10⁷ unopsonized IE or IE opsonized with pooled immune patient serum (IE-PPS) for 24 hr followed by preparation of nuclear and cytoplasmic extracts. (A) Extracts were analysed by immunoblotting using antibodies detecting the p50 and p65 NF-κB subunits. Blots were re-probed using antibodies to GAPDH and TATA-binding protein (TBP) to assess the purity of cytoplasmic (C) and nuclear (N) fractions respectively. (B) IFN-γ-primed MDM were incubated with IE-PPS or IE or incubated in medium alone (MDM) for 24 hr, and nuclear extracts prepared as in (A). Binding of nuclear proteins to an NF-κB specific oligonucleotide was analysed using EMSA as described in Methods. Saturable binding was indicated by competition using 100-fold excess of unlabelled probe (lanes 4,6), specificity of binding by lack of competition using a mutant oligonucleotide probe (lanes 5,7) and the presence of p50 and p65 in DNA binding complexes confirmed by incubation with anti p50 and p65 or control monoclonal antibodies (lanes 8–10). The positions of complexes containing p50/p65 heterodimer are labelled NF-κB.