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Fig. 8 Both zebrafish PKR and PKZ phosphorylate yeast eIF2α. (A) Plasmids expressing full-length human (hs) PKR or zebrafish (dr) PKZ or two different alleles of PKR, under the control of a galactose-inducible promoter, were introduced into S. cerevisiae strains H2557 (wild-type eIF2α, upper plates) and J223 (eIF2α-S51A, lower plates) as indicated. After two rounds of single colony purification, the transformants were streaked out simultaneously on SD-ura (non-inducing conditions, left) or SGal-ura (inducing conditions, right) medium and grown for three days (SD-ura) or four days (SGal-ura). (B) Western blot analyses of extracts from strain H2557 transformed with vector, HsPKR, DrPKR or DrPKZ. SDS-PAGE was used to separate 4 μg of WCEs which were blotted onto nitrocellulose membranes. Tagged PKR and PKZ were detected using anti-Flag-tag antibody (upper panel). eIF2α phosphorylated on Ser51 and total eIF2α are shown in middle and bottom panels, respectively.