FIGURE 3.

Effects of H₂O₂ on mRNA translation and protein synthesis. A, HEK293 cells and immortalized wild-type mouse embryonic fibroblasts (MEFs) were exposed to 0-500 μm H₂O₂ for 1 h. Whole cell extracts were blotted for 4EBP1, phospho-rpS6, phospho-eIF2α, total eIF2α, phospho-eEF2, and total eEF2. Hypophosphorylated (α) and phosphorylated (β and γ) forms of 4EBP1 are indicated. Levels of total eIF2α and eEF2 proteins were examined for sample loading and protein stability using the same lysates run on separate gels. Changes in eIF2α phosphorylation (based on Image J analysis) compared with 0 μm H₂O₂ are shown. B, protein synthesis in H₂O₂-treated (1 h) MEFs with or without 2 h BME (100 μm) preconditioning. BME (100 μm) was present during the 1-h protein synthesis. ^**, p < 0.01. C, Western blotting for total 4EBP1 protein and rpS6 phospho-Ser^235/236 in H₂O₂-treated (1 h) TSC2^+/+ and TSC2^-/- MEFs. Changes in rpS6 phosphorylation compared with 0 μm H₂O₂ are indicated. D, protein synthesis in TSC2^+/+ and TSC2^-/- MEFs treated with 100 μm H₂O₂ (1 h). [³⁵ S]Methionine labeling was carried out in the presence of 100 μm H₂O₂. Base-line protein synthesis was similar in TSC2^+/+ and TSC2^-/- cells.