Fig. 3.

Dephosphorylation of eIF2α in vitro by purified GLC7. (A) Purified yeast eIF2 complexes containing WT or the indicated mutant forms of eIF2γ were incubated with FLAG-PKR immobilized onto M2-conjugated agarose and ATP to phosphorylate eIF2α on Ser51. Following incubation of the phosphorylated eIF2 complexes with purified GST-GLC7-FLAG for the indicated times (min), the reaction products were subjected to immunoblot analysis using specific polyclonal antibodies to detect eIF2α–P and total eIF2α. Monoclonal antibodies against the His-tag were used to detect eIF2γ, and polyclonal antibodies against GST were used to detect GLC7. (B) The ratio of eIF2α–P to total eIF2α at each time point was quantified from three independent experiments and normalized for each eIF2 complex to the value at time 0.