Fig. 5.

The eIF2α pathway mediates the translational arrest of D2 synthesis during ER stress. D2 protein synthesis is decreased in ER-stressed cells. A, HEK-293 cells transiently expressing a FLAG-tagged CysD2 were treated with vehicle or 300 nm thapsigargin (Th) for 1 h. After this incubation period, [³⁵S]methionine was added to the incubation media for an additional 1 h. Total treatment time with thapsigargin was 2 h. CysD2 was immunoprecipitated and resolved on a 12% SDS-PAGE. Image shown represents a 2-d exposure to radiographic film. In panel B, D2 activity levels of MSTO-211H cells electroporated with either a control or a eIF2a-encoding vector with a inactivating S52A mutation. Cells were exposed to either vehicle or 25 nm thapsigargin for 1 h. C, Top panel, phospho and total eIF2a protein levels; in lower graph, CHOP mRNA levels of MSTO-211H cells from panel B. All values are displayed as ± sem and data were normalized to untreated group and are representative of two independent experiments. B, *, P < 0.01 vs. vehicle-treated group. C, *, P < 0.01 vs. vehicle-; **, P < 0.01 vs. S52A Th-treated group by one-way ANOVA. When two or more groups are compared with vehicle-treated group, Dunnett's posttest was used. WT, Wild type.