Fig. 2: H5N1 virus induces E-cadherin, occludin, and claudin-1 degradation. | Communications Biology

Fig. 2: H5N1 virus induces E-cadherin, occludin, and claudin-1 degradation.

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

Fig. 2

a H5N1 viruses accelerate the turnover of intercellular junction proteins. A549 cells infected with 1 MOI of H5N1 viruses were incubated for 24 h in the absence or presence of MG132 (10 μM). Cell lysates were analyzed by Western blot for the levels of E-cadherin, occludin, claudin-1, ZO-1, and the viral NP and NS1 proteins with their specific antibodies. b H5N1 viruses shorten the half-life of intercellular junction proteins. A549 cells were left uninfected or infected with H5N1 viruses (1 MOI). After incubation for 12 h, CHX (10 μM) was added and incubated for the indicated lengths of time. Cell lysates were prepared and analyzed for E-cadherin, occludin, claudin-1, and NP by Western blot. c Quantification of relative E-cadherin levels (from b). d Quantification of relative Occludin levels (from b). e H5N1 viruses shorten the half-life of occludin. A549 cells were transiently transfected with the pcDNA3.1 expression vector encoding the FLAG-occludin gene. After incubation for 24 h, the cells were then left uninfected or infected with the H5N1 virus (1 MOI). After incubation for 12 h, CHX (10 μM) was added and incubated for the indicated lengths of time. Cells were harvested and analyzed for occludin by Western blot. The density of bands was analyzed using NIH Image-J software and normalized by the arbitrary units of β-actin. f Quantification of relative Occludin levels (from e). g Quantification of relative Claudin-I levels (from b). Data presented in c, d, f, and g are the mean ± SD from one of three experiments in triplicate. h, i H5N1 virus induces occludin ubiquitination. A549 cells were transiently transfected with an HA-ubiquitin expression vector alone (h) or plus the pcDNA3.1 expression vector encoding the FLAG-occludin gene (i). After incubation for 24 h, the cells were left uninfected or infected with the H5N1 virus (1 MOI). After incubation for another 24 h, cell lysates were prepared and immunoprecipitated with an antibody against occludin (h) or FLAG (i). Whole-cell lysates (WCL) and immunoprecipitates were analyzed with antibodies against ubiquitin, NP, occludin, and β-actin. The data represent one of three independent experiments. *p < 0.05, **p < 0.01.

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