Figure 2. Overexpression of PARK2 inhibits localization of the nuclear NF-κB for inflammation.

A-C. After infection with the indicated shRNAs, Primary and immortalized (BEAS-2B) human bronchial epithelial cell lysates were fixed for immunofluorescence assay or blotted with the indicated antibodies. (A) Representative images of the cells with indicated localization of the nuclear NF-κB (white arrows). Cells were fixed and stained with DAPI. TNF-α-treated cells were shown as positive controls. Scale bar, 20 μm. (B) Analysis of numerical nuclear NF-κB events. 100 cells were counted in each experiment. **, p < 0.01 and ***,p < 0.001 by one-way ANOVA. (C) Immunoblot analysis with indicated antibodies after nuclear fractionation. D. After transfection with the indicated plasmids, Beas-2B cells were selected by G418 to select stable transfectants, cells were treated with TNF-α and cells lysates were fixed for immunofluorescence assay or cell lysates were analyzed immunobloting. Representative images of the cells with indicated localization of the nuclear NF-κB (white arrows). Cells were fixed and stained with DAPI. TNF-α-treated cells were shown as negative controls. Scale bar, 20 μm. Quantification of the nuclear NF-κB localization. Results are shown as means (±SEM), and at least three experiments were performed for all experiments. *, p < 0.05, **, p < 0.01 and ***, p < 0.001 by one-way ANOVA. E. After nuclear fractionation, NF-κB, Lamin B1 and β–tubulin expression was measured by immunoblot. Lamin B1, nuclear marker; β–tubulin, cytosol marker. F. Cells were stained with DCF-DA, fixed and analyzed by immunofluorescence. G. Cells were harvested and analyzed by immunobloting. H. Cells were treated with the indicated chemicals, MK 2206 (an allosteric Akt inhibitor) or NAC (ROS scavenger). After nuclear fractionation, NF-κB, Lamin B1 and β–tubulin expression was measured by immunoblot. Lamin B1, nuclear marker; β–tubulin, cytosol marker.