Figure 2

p38 MAPK-dependent NFκB-p65 activation in HAdV-19 infected keratocytes. Cells were incubated with the p38 MAPK inhibitor SB203580 (5 or 10 μM) prior to HAdV-19 or mock infection, and lysates prepared 30 min after infection. (A) NFκB-p65 and IκB phosphorylation increased with virus compared to mock infection, and were reduced in cells pre-treated with SB203580 in dose-dependent fashion. Densitometry values for the phosphorylated protein (normalized to the corresponding actin levels) are shown above each lane. (B) Immunoprecipitation assay reveals association of p38 MAPK with NFκB-p65 in virus infected (V) but not in mock (M) infected cells. This association was blocked by the p38 MAPK inhibitor SB203580 (SB) but not by the JNK inhibitor SP600125 (SP). Isotype control did not immunoprecipitate any NFκB-p65. (C) NFκB-p65 activation in HAdV-19 infection analyzed by confocal microscopy. The left column shows DAPI staining for nuclei (blue), the middle column for p65 (yellow), and the right column a merging of the left 2 rows. (c) Mock infected keratocytes show mostly cytoplasmic localization of NFκB-p65. (f) HAdV-19 infected keratocytes at 20 min post-infection show nuclear localization of NFκB-p65. (i) Nuclear translocation of NFκB-p65 was reduced in the presence of 10 μM, and (l) completely blocked with 20 μM SB203580. Bottom row (m, n, o), represent isotype control. (D) Electromobility shift assay showing NFκB-p65 binding to IL-8 promoter in HAdV-19 infected keratocytes. Extracts from HAdV-19 infected cell nuclei show more binding to NFκB-specific IL-8 probe (lane 2) as compared to nuclear protein from mock infected cells (lane 6) or when pretreated with SB203580 (lane 10). Binding specificity of the probe is shown with 100 molar excess of unlabelled probe (lanes 3 and 7). SB203580 (SB) blocked NFκB binding in virus infected nuclear extracts (lane 10). Dose dependent supershifts using increasing amounts of NFκB-p65 antibody are shown in virus infected nuclear extracts (lanes 4 and 5). No shifts were observed in nuclear extracts from mock infected cells (lanes 8 and 9) or SB203580 treated cells (lane 11).