Fig. 2

Activation of the PERK pathway by TMUV infection. a, Phosphorylation of eIF2α was detected by western blotting. Mock- or TMUV-infected BHK-21 cells were lysed and harvested at the indicated time points. Phosphorylated or total eIF2α was analysed by western blotting using corresponding antibodies. Tubulin was used as an internal control. The intensities of bands were determined using IMAGE J software, and the data represent ratios of TMUV-infected cells to mock-infected cells at the indicated time points. b, Inhibition of eIF2α phosphorylation in BHK-21 cells by treatment with the PERK inhibitor GSK2606414. Cells were harvested at 24 h post-infection and subjected to western blotting. The intensities of phospho-eIF2α and eIF2α were determined using IMAGE J software, and the results are shown as ratios of TMUV-infected cells to mock-infected cells. c, BHK-21 cells were infected with TMUV at an MOI of 3 and treated with 1 μM GSK2606414 or the corresponding level of DMSO or were not treated. Untreated mock-infected BHK-21 cells were used as a control. Twenty-four hours post-infection, cell viability was assessed using the CCK-8 kit. The data are expressed as the means±SD of results from three independent experiments. d, Real-time RT-PCR analysis of components of the PERK pathway during a time course of TMUV infection. The Y axis shows the fold change of target gene expression in TMUV-infected cells, as determined using the comparative CT method. The data are expressed as the means±SD of results from three independent experiments. * p<0.05 vs. mock-infected cells. e, Protein levels of ATF4, GADD34 and CHOP were examined by western blotting. The intensities of bands were determined using IMAGE J software, and the data represent ratios of TMUV-infected cells to mock-infected cells at the indicated time points