Fig. 6: Role of TAK1 in degrading junction proteins.

a H5N1 viruses induce TAK1 phosphorylation. A549, MDCK, and NL20 cells were infected with various MOI of the H5N1 viruses for 24 h or with the H5N1 viruses (1 MOI) for the indicated lengths of time. Cell lysates were analyzed for the levels of TAK1 phosphorylation, NP, NS1, and β-actin by Western blot. b 5Z blocks H5N1 virus-induced Itch expression and junction protein downregulation. A549 cells were left uninfected or infected with 1 MOI of H5N1 viruses. After incubation for 12 h, 5Z (5 μM) was added and then incubated for another 12 h. Cell lysates were analyzed by Western blot for TAK1, ERK, JNK, p38, and p65 phosphorylation and for the levels of junction proteins with their specific antibodies. The density of bands was analyzed using NIH Image-J software and normalized by the arbitrary units of β-actin. Data are the mean ± SD of three experiments. *p < 0.05, **p < 0.01. c 5Z blocks H5N1 virus-induced disruption of intercellular junction structure. A549 cells seeded on coverslips were treated as above. Cells were immunostained with antibodies against occludin and claudin-1 and visualized under a confocal microscope. d Quantification of fluorescence signals. The monolayers immunostained for occludin and claudin-1 in c 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 5Z blocks the H5N1 virus-induced decrease of electronic resistance. A549 cells seeded in Transwell inserts were left uninfected or infected with H5N1 viruses. After incubation for 12 h, 5Z (5 μM) was added and then incubated for another 12 h. TEER was measured at the indicated times. The results represent the mean ± SD of three independent experiments. **p < 0.01.