A Novel Peptide Derived from Human Pancreatitis-Associated Protein Inhibits Inflammation In Vivo and In Vitro and Blocks NF-Kappa B Signaling Pathway
Figure 9
Effect of PAPep on nuclear translocation of NF-κB in RAW264.7 cells.
(A) The intracellular location of NF-κB p65 was determined in RAW264.7 cells by immunofluorescence using an anti-NF-κB p65 antibody with Alexa Fluor 555 labelling (red fluorescence), and the nuclei were counterstained with DAPI (blue fluorescence). (a) Untreated cells exhibit the localization of NF-κB in the cytoplasm. Cells stimulated with 100 ng/ml LPS (b) and 50 µM PAPS(c) display a significant increase in the translocation of NF-κB into the nucleus. (d) Stimulated cells in the presence of 50 µM PAPep maintained predominantly cytoplasmic NF-κB immunostaining, indicating inhibition of NF-κB translocation. Results are displayed individually or as merged images (confocal fluorescence microscopy). Data represent one of three experiments with similar results. Scale bars, 10 µm. (B) Nuclear NF-κB was quantitated by visual fluorescent microscopy. The number of cells with p65 nuclear translocation in six random fields were counted in a masked fashion and expressed as a percentage of the number of translocated cells in comparison to that of total cells. ##, P<0.01 compared with control group, **, P<0.01 compared with LPS group.