Phosphorylation of eIF2α upon coumermycin-induced GyrB-PKR dimerization. (A) Schematic diagrams showing the fusion of PKR kinase domain with coumermycin-binding domain of GyrB. Application of coumermycin (C. mycin) induces the dimerization of the PKR fusion proteins and activates PKR, triggering eIF2α phosphorylation and subsequent de novo protein synthesis inhibition. (B) Representative blots showing C.mycin-induced phosphorylation of eIF2α on Ser51 in Xenopus embryos expressing GyrB-PKR. Embryos were treated with various concentrations of coumermycin for 8 hours, harvested and lysed. Western blotting was performed using specific antibodies as indicated. The blots were also probed with anti-eIF2α and anti-tubulin antibody for loading controls. (C) Quantification of eIF2α phosphorylation with different C.mycin concentrations. (D) Time course of eIF2α phosphorylation induced by 1 μM Coumermycin. (E) Time course of eIF2α phosphorylation upon 1 μM C.mycin treatment and withdrawal. Embryos were treated with Coumermycin for 2 hours and washed with culture medium without C.mycin (F) Quantification of eIF2α phosphorylation with or without C.mycin at different time points. Arrow indicated the time point of withdrawing C.mycin. Note the eIF2α phosphorylation level goes back to the baseline within 8 hours. Multiple blots were quantified (N = 6), and eIF2α-P signals at various time points were normalized to that at "0" hour.