Fig. 4: Role of p38 in H5N1 virus-induced Itch expression and degradation of junction proteins. | Communications Biology

Fig. 4: Role of p38 in H5N1 virus-induced Itch expression and degradation of junction proteins.

From: H5N1 infection impairs the alveolar epithelial barrier through intercellular junction proteins via Itch-mediated proteasomal degradation

Fig. 4

a H5N1 viruses induce p38 phosphorylation. A549 cells were infected with the indicated MOI of H5N1 viruses for 24 h or infected with 1 MOI of H5N1 viruses for the indicated lengths of time. Cell lysates were analyzed for the levels of p38 phosphorylation, NP, and β-actin by Western blot. b SB202190 blocks H5N1 virus-induced Itch expression and junction protein downregulation. A549 cells were left uninfected or infected with the H5N1 virus (1 MOI). After incubation for 12 hr, SB202190 (10 μM) was added and incubated for another 12 h. Cell lysates were prepared and analyzed for the levels of p38 phosphorylation, Itch, and junction proteins by Western blot with the indicated antibodies. The density of bands was analyzed using NIH Image-J software and normalized by the arbitrary units of total p38 or β-actin. Data represent the mean ± SD of three experiments. *p < 0.05, **p < 0.01. c SB202190 blocks H5N1 virus-induced disruption of intercellular junction structure. A549 cells were left uninfected or infected with H5N1 viruses (1 MOI). The cells were then incubated in the absence or presence of SB202190 (10 μM) for 24 h. The cells were fixed and stained with the antibodies against occludin or claudin-1. d Quantification of fluorescence signals. The monolayers immunostained for occludin and claudin-1 were analyzed for immunofluorescence intensity by using Image J software. The fluorescent signals of intercellular junctions were quantified and plotted as bar graphs. Data represent the mean ± SD of five random fields (40X) from one of three independent experiments with similar results. e SB202190 prevents the H5N1 virus-induced decrease of electronic resistance. A549 cells seeded in the Transwell inserts were left uninfected or infected with H5N1 viruses in the absence or presence of SB202190 (10 μM). TEER was measured at the indicated times. The results represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01.

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