Fig. 5: Role of ERK in H5N1 virus-induced Itch expression and the degradation of junction proteins.

a H5N1 viruses induce ERK phosphorylation. A549 cells were infected with the indicated MOI of H5N1 viruses for 24 h or infected with H5N1 viruses (1 MOI) for the indicated lengths of time. ERK1/2 phosphorylation was analyzed by Western blot. b U0126 blocks H5N1 virus-induced Itch expression and junction protein downregulation. A549 cells were left uninfected or infected with H5N1 viruses (1 MOI). After incubation for 12 h, U0126 (10 μM) was added and incubated for another 12 h. Cell lysates were analyzed for the levels of ERK phosphorylation, Itch, and junction proteins by Western blot with the indicated antibodies. c U0126 blocks H5N1 virus-induced disruption of intercellular junction structure. A549 cells were infected with H5N1 viruses (1 MOI). After incubation for 12 h, the cells were incubated in the absence or presence of U0126 (10 mM) for another 12 h. The cells were fixed and then stained with antibodies against occludin and claudin-1. d Quantification of fluorescence signals. The monolayers immunostained for occludin and ZO-1 in c were analyzed for immunofluorescence intensity by using Image J software. The fluorescent signals of intercellular junction proteins were quantified and plotted as bar graphs. The data represent the mean ± SD of five random fields (40X) from one of three independent experiments with similar results. e U0126 blocks the H5N1 virus-induced decrease of electronic resistance. A549 cells seeded in Transwell inserts were left uninfected or infected with H5N1 viruses in the absence or presence of U0126 (10 μM). TEER was measured at the indicated lengths of time. The results represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01. f H5N1 viruses induce JNK phosphorylation. A549 cells were infected with the indicated MOI of H5N1 viruses for 24 h or infected with H5N1 viruses (1 MOI) for the indicated lengths of time. JNK phosphorylation was analyzed by Western blot. g SP600125 does not block H5N1 virus-induced downregulation of junction proteins. A549 cells were left uninfected or infected with H5N1 viruses (1 MOI). After incubation for 12 h, U0126 (10 μM) was added and incubated for another 12 h. Cell lysates were analyzed for the levels of ERK 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 ERK, JNK, or β-actin. Data are the mean ± SD of three experiments. *p < 0.05, **p < 0.01.