NBD peptide selectively blocks canonical NF-κB activation. (A) NBD peptide blocks CD40L-induced IκBα phosphorylation. Monocyte-derived DCs were preincubated with either NBD peptide or MUT peptide for 2 hours. Subsequently, cells were stimulated with CD40L for 30 minutes, extensively washed, and lysed in sample buffer. Cell lysates were analyzed by Western blotting and densitometry was performed. One representative experiment of 3 is shown; densitometry includes data from all experiments (*P < .05). (B) NBD peptide completely blocks nuclear translocation of RelB following LPS stimulation, whereas CD40L-induced RelB translocation is only marginally affected. Series of confocal images of monocyte-derived DCs stimulated for 4 hours with LPS or CD40L in the presence or absence of NBD/MUT peptides or controls. Cells were centrifuged onto glass slides, fixed in cold acetone, and stained for RelB expression. Nuclei were stained with Hoechst, and cells were analyzed by scanning the entire cell using a confocal laser microscope. In the displayed overlay pictures, RelB nuclear translocation can be evaluated. Representative pictures from one experiment are shown. Results are representative of 3 independent experiments. See “Materials and methods, Immunofluorescence staining” for image acquisition information.