Figure 6

ER stress inhibits protein synthesis through phosphorylation of eIF2α in EL4 stable cell lines. (A) Decreased overall rate of protein synthesis upon ER stress. EL4 stable cell lines were deprived of glucose, treated with 0.25 mM of palmitate, 100 μg/ml of cycloheximide or cultured under control conditions for 19 hours. [³H]Leucine (10 μCi/mL) was added during the last hour. The rate of protein synthesis was measured by [³H]leucine incorporation. The results are expressed as the % of [³H]leucine incorporation per cell relative to control cells (dotted line) in EL4/HEL-Cyto-SIINFEKL (blue) and EL4/HEL-ER-SIINFEKL (red) cells. Bars depict the mean and SD of one representative experiment performed in triplicate. Differences between untreated and treated cells are all significant (P < 0.05). (B) Phosphorylation of eIF2α. EL4 cells were deprived of glucose or treated with 0.25 mM of palmitate for different durations over a 24-h period. Total cell lysates were immunoblotted against phosphorylated eIF2α (Ser51) or total eIF2α. α-tubulin was used as loading control. One representative experiment out of three is shown.