Stimulation of DCs with CCL19 induces activation and translocation of NFκB to the nucleus. (A) Determination of NFκB DNA-binding activity in total cell extracts obtained from DCs that were left unstimulated (–) or stimulated (+) with CCL19 for 30 minutes. Extracts were used to perform EMSA in the absence (–) or presence (+) of a 100-fold molar excess of unlabeled competitor (Comp) oligonucleotides. A representative experiment out of 2 performed is shown. (B) Stimulation of DCs with CCL19 induces translocation of NFκB to the nucleus. DCs were transfected with either pEGFP-C1 vector (GFP-vector) or pEGFP-p65 (GFP-p65). Eighteen hours after transfection, DCs were washed with RPMI and then either left unstimulated (–) or stimulated for 45 minutes with CCL19 or TNF-α used as positive control. The cells were then plated onto polylysine-coated coverslips, fixed, permeabilized, and the nucleus stained with Hoechst 33342 (not shown). The figure show Nomarski optics and GFP protein staining analyzed using the FITC fluorescence channel. Hoechst 33342 staining marked the position of the nucleus in all the cases (not shown). The figure was viewed with a Nikon Diaphot microscope. A representative experiment out of 3 performed is shown.