Article Title: Investigation of fascin1, a marker of mature dendritic cells, reveals a New role for IL-6 signaling in chemotaxis
Figure Lengend Snippet: IL-6Rα signaling is critical for CCR7 internalization and CCL19-induced ERK1/2 phosphorylation. A , Effects of the blockage of IL-6 signaling on CCR7 internalization. WT BMDCs were stimulated with CCL19 for 5 min in the presence of either control (a) or neutralizing antibody (b), and then surface CCR7 expression was determined by flow cytometry. Surface expression of CCR7 was examined with CD86 + BMDCs before (-CCL19) and after CCL19 addition (+CCL19). Note that CCR7 internalization was blocked in the absence of a neutralizing antibody to IL-6Rα. B , Effects of IL-6 signaling on CCR7 internalization of IL-6Rα KO BMDCs. IL-6Rα KO BMDCs were stimulated with CCL19 in the absence (a) or presence of sIL-6Rα (soluble IL-6Rα) and IL-6 (b). Note that IL-6Rα KO BMDCs showed impaired CCR7 internalization (a), which was rescued by the addition of sIL-6Rα and IL-6 (b). C , Inhibition of CCR7 internalization by the blockage of IL-6 signaling in HEK293T epithelial cells. HEK293T cells stably expressing mouse CCR7-GFP were stimulated with CCL19 in the presence of either control or neutralizing antibody against IL-6Rα. a-d, fluorescence images of CCR7-GFP in control cells (a b) or IL-6Rα neutralizing antibody-treated cells (c d) before (a c) or after addition of CCL19 (b d). The same cells were counter stained with an antibody specific to mouse CCR7 to detect only surface CCR7 (e-h) in control (e f) or IL-6Rα neutralizing antibody-treated cells (g h) before (e g) or after (f h) addition of CCL19. D , Quantitative measurements of surface CCR7 immunofluorescence (panels e-h of C) at cell-to-cell contacts. In contrast to the control, the addition of a neutralizing antibody against IL-6Rα blocked internalization of CCR7. E , Effects of the blockage of IL-6 signaling on CCL19-induced ERK1/2 phosphorylation of WT BMDCs. WT BMDCs were stimulated with CCL19 in the presence of a control or neutralizing antibody, then ERK1/2 phosphorylation levels were determined at 0, 5, 15 and 30min after CCL19 addition using Western blotting with a phosphospecific ERK1/2 antibody. For normalization, the same membranes were reblotted with a pan ERK1/2 antibody. The ratios of phosphoERK1/2 to total ERK1/2 are indicated below the figure.
Article Snippet: To restore IL-6 signaling of IL-6Rα KO DCs, human IL-6 (15ng/ml, 206-IL, R & D Systems) and soluble IL-6Rα (sIL-6Rα, 25ng/ml, 227-SR, R & D Systems) were added 1hr after LPS addition.
Techniques: Expressing, Flow Cytometry, Inhibition, Stable Transfection, Fluorescence, Staining, Immunofluorescence, Western Blot