Figure 5.

PERK kinase activity stimulates DLL4 IRES-mediated translation in a phosphorylated eIF2α–dependent manner. (A) Linear schematic representation of PERK and PERK-LZ showing the locations of the major functional domains (SP = Signal Peptide, L zip = Leucine Zipper, HA = HA Tag). Numbers indicate amino acid positions. (B) Western blot analysis of doxycycline-induced-PERK-LZ (HA) expression and eIF2α phosphorylation after 48 h treatment with increasing amounts of doxycycline. (C) Relative IRES activities in PERK-LZ expressing HeLa cells treated with 1 µg/mL doxycycline /control after transfection of EMCV or DLL4 bicistronic vectors. Means ± SEM are shown, *** p < 0.001. (D) Western blot analysis of ATF4, total and phosphorylated eIF2α, as well as total PERK in HeLa cells, after transfection with ATF4 or scramble (Scr) siRNA and treatment with DTT. β-ACTIN was used as a loading control. (E) Relative IRES activities in HeLa cells treated with DTT/control, after cotransfection with the EMCV or DLL4 bicistronic vectors and with either siRNA specific for ATF4 or control siRNA (Scr). Results represent the means of three independent experiments ± SEM. (F) Wild-type (WT) and eIF2αS51A MEFs transfected with the bicistronic LucR-IRES-LucF vectors and treated with increasing concentrations of DTT. ER stress induction was verified by monitoring eIF2α phosphorylation by western blot and cytoplasmic XBP1 splicing by RT-PCR. (G) Relative IRES activities were determined as previously described in WT (left) and eIF2αS51A (right) MEFs. Results represent the means of three independent experiments ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.