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. 2014 Nov;88(21):12752–12764. doi: 10.1128/JVI.02138-14

FIG 6.

FIG 6

JNK and Akt are involved in the antiapoptotic function of IRE1α. (a) JNK hyperphosphorylation and Akt hypophosphorylation in IRE1 knockdown cells infected with IBV. H1299 cells were transfected with siIRE1 or nontarget siRNA before being infected with IBV and harvested at the indicated time points. Western blot analysis was performed using antibodies against IRE1α, IBV N, PARP, phosphor-JNK, total JNK, phosphor-Akt, and total Akt. β-Actin was included as a loading control. The percentage of PARP cleavage was calculated as for Fig. 3a. The percentage of JNK or Akt phosphorylation was calculated as the band intensity of phosphorylated JNK or phosphorylated Akt divided by the band intensity of the corresponding total JNK or total Akt, respectively. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. The asterisks indicate significant differences between the indicated samples and the siNC-transfected samples at the same time point (*, P < 0.05; **, P < 0.01). (b) Akt protects cells from IBV-induced apoptosis. H1299 cells were transfected with pcDNA3.1 or pcDNA3.1-myr-AKT1. At 24 h posttransfection, the cells were infected with IBV (MOI, ∼2) or mock infected. Cells were harvested at the indicated time points and subjected to Western blot analysis using antibodies against IBV N, PARP, phosphor-Akt, and total Akt. β-Actin was included as a loading control. The percentage of PARP cleavage was calculated as for Fig. 3a. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. The asterisks indicate significant differences between the indicated samples and the pcDNA3.1-transfected samples at the same time point (**, P < 0.01). (c) JNK is required for IBV-induced apoptosis. H1299 cells were transfected with siJNK1/2 or siEGFP before being infected with IBV or mock infected. Cells were harvested at the indicated time points and subjected to Western blot analysis using antibodies against IBV N, PARP, phosphor-JNK, and total JNK. β-Actin was included as a loading control. The percentage of PARP cleavage was calculated as for Fig. 3a. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. The asterisks indicate significant differences between the indicated samples and the siEGFP-transfected samples at the same time point (*, P < 0.05; **, P < 0.01).