Phosphorylation of eIF2α is required for increased <i>TMEFF2</i> translation in response to ER stress.

<p><b>A)</b> Mouse embryonic fibroblast (MEF) cells expressing the wild type (S/S) or mutant (Ser51 to Ala; A/A) eIF2 proteins were treated with the indicated concentrations of clotrimazole (CLT) or thapsigargin (TG) and protein lysates from these cells were analyzed by western blot to determine the extent of eIF2α-P. Total eIF2α was used as loading control. <b>B)</b> Luciferase activity demonstrated by the pTM1234-Gluc reporter construct in wt (S/S) or mutant (A/A) MEFs in the absence or presence of the indicated concentrations of thapsigargin. Luciferase activity was normalized to the luciferase activity of the reporter expressed in cells grown in the absence of thapsigargin, which was set to 1. Data shown are mean ± S.D. of at least three independent experiments with multiple replicates. *, <i>p</i><0.05, and **, <i>p</i><0.01. <b>C)</b> Western blot analysis demonstrating increased ATF4 expression as a result of thapsigargin treatment in MEF cells carrying the wt form of the eIF2α subunit (MEF S/S) but not the mutant form mutant (MEF A/A). β-tubulin was used as a loading control.