Figure 5
Effect of eIF2α phosphorylation on membrane transporter expression under ER stress preconditioning. WT, stable cell line transfected with wild-type eIF2α plasmid; SA, stable cell line transfected with mutant eIF2α-SA plasmid. (A) Synthetic siRNA-mediated knockdown of endogenous Eif2α. Western blots of WT and mutant SA cell lines transfected with siRNA against endogenous Eif2α were analyzed using anti-eIF2α antibody probes. NS, non-silencing. Ei, Eif2α siRNA. Representative images of 3 samples are shown. (B) Effect of mutant non-phosphorylatable eIF2α on ATF4, phospho-eIF2α, and eIF2α expression. Western blots of untreated endogenous Eif2α-knockdown WT- or SA-expressing cell lines or pretreated with 0.3 μg/ml TPG for 16 h were analyzed using the indicated antibody probes. Cropped blots shown; all gels were run under the same experimental conditions. Representative images of 3 samples are shown. (C–E) Effect of eIF2α phosphorylation on the expression of membrane transporter (C), Snhg1 (D) or Atf4 (E) mRNA was analyzed by RT-qPCR. Total RNA was extracted from untreated cells or following treatment with 0.3 μg/ml TPG for 16 h. The histogram depicts the indicated mRNA normalized to Actb mRNA. Values shown are the means ± SE of 4 separate experiments. **, ***Significantly different from TPG-untreated cells by a one-way ANOVA (**p < 0.01, ***p < 0.001). ##, ###Significantly different from TPG-treated NS siRNA-transfectants by a one-way Welch’s t-test (##p < 0.01, ###p < 0.001).
