Fig. 5

Fig. 5

Effects of DrPKZ-A and DrPKZ-B on eIF2α phosphorylation. Lanes 1–7 represent cells co-transfected with the following recombinant vectors: pcDNA3.1a, PKZ-A-3.1a, PKZ-B-3.1a, PKZ-A ΔN-3.1a, PKZ-B ΔN-3.1a, PKZ-A ΔID-3.1a, PKZ-B ΔID-3.1a, and eIF2α-Myc. (A) The phosphorylation levels of eIF2α are shown in WB1, which was blotted with a rabbit anti-phospho-eIF2α (Ser51) antibody. Western blotting analysis for eIF2α and actin is shown in WB2 and WB3, which were blotted with rabbit anti-Myc and rabbit anti-actin antibodies, respectively. (B) Western blotting analysis to determine the expression of all recombinant vectors with mouse anti-V5 antibody. As indicated by the arrows, all recombinant vectors were expressed normally at the predicted molecular weights. (C) Quantification of the phosphorylated eIF2α levels versus actin levels. Compared with the control group (cells transfected with an empty vector) (column 1), the DrPKZ-A and DrPKZ-B-expressing cells display increases in the phosphorylation levels of eIF2α of approximately 3.5-fold and 2.6-fold, respectively (columns 2 and 3). DrPKZ-A ΔN and DrPKZ-B ΔN were less effective at phosphorylating eIF2α. Compared with the control group, these two truncated mutants elevate the phosphorylation levels of eIF2α by about 2.5-fold and 1.5-fold (columns 4 and 5). DrPKZ-A ΔID and DrPKZ-B ΔID lost nearly all of their kinase activity to eIF2α (columns 6 and 7). Data are presented as the means α S.D. (n = 3 replicates). * indicates a statistically significant difference relative to the pcDNA3.1a control (P < 0.01). ** indicates a statistically significant difference between DrPKZ-A and DrPKZ-B, and ++ indicates a statistically significant difference between DrPKZ-A ΔN and DrPKZ-B ΔN.