Role of the NF-κB pathway in MoDC maturation.

(A) LPS induces the degradation of Iκ-Bα in MoDCs. Day-7 immature MoDCs were exposed to LPS (100 ng/mL) for variable lengths of time (as shown), after which samples were removed and analyzed by Western blotting with an antibody directed against Iκ-Bα. The blot shows the degradation of Iκ-Bα induced in MoDCs by the addition of LPS. The lower panel shows the blot probed for tubulin to demonstrate equal loading of samples. Similar results were obtained in 3 separate experiments. (B) SN50 peptide inhibits the LPS-induced nuclear translocation of NF-κB. Day-7 immature MoDCs were stimulated with LPS (100 ng/mL) after preincubation with SN50 or control SN50M peptide (50 μg/mL), after which nuclear extracts were prepared. Oligonucleotides (containing the consensus binding sequence for NF-κB) bound to agarose beads were used to pull down nuclear NF-κB. The resulting samples were analyzed by SDS/PAGE and immunoblotting using a polyclonal anti-p65 NF-κB antibody. LPS can be seen to induce the nuclear translocation of NF-κB within 30 minutes of its addition to MoDCs. This is prevented by the addition of the SN50 peptide. The control peptide had no such effect.