FIG 4.

Expression of ASFV MGF110-7L enhances eIF2α phosphorylation through upregulation of PERK and PKR activity. (A) Diagrams of four major stress pathways leading to eIF2α phosphorylation by different kinases, PERK, PKR, GCN2, and HRI. (B and C) 3D4/21 and PK-15 cells were transfected with the MGF110-7L-Flag-expressing vector with an increasing dose (0.5, 1.0, 2.0 μg) or an empty Flag vector (2.0 μg) for 24 h. As a positive control, the cells were treated with TG (1 μM) for 6 h. The whole-cell lysates were then obtained and immunolabeled using the indicated antibodies (left). Densitometry analysis of these protein bands in MGF110-7L-transfected cells compared to empty vector-transfected cells was performed using ImageJ (right). (D) 3D4/21 cells were treated with the indicated concentrations (1, 5, and 10 μM) of GSK2606414 and C16 for 24 h, and cell viability was measured using a CCK-8 assay. (E and F) 3D4/21 cells were transfected with an empty Flag vector or a MGF110-7L-Flag-expressing vector (2.0 μg), with or without GSK2606414 (10 μM) or C16 (1 μM) as indicated for 24 h before cell lysate samples were obtained. Lysates were analyzed via immunoblotting with the indicated antibodies. The relative levels of p-PERK, p-PKR, or p-eIF2α in each sample after normalizing to the corresponding total PERK, total PKR, or total p-eIF2α was determined using ImageJ software and plotted in bar graphs. Data in panels B to D show means ± the SD of the results of three independent experiments. **, P < 0.01, ns, not significant.