Figure 6.

Role of eEF2K in eIF2α-Independent Translational Inhibition

(A) Western blot analysis was performed with lysate prepared from eEF2K^−/− and eIF2α^S51A/eEF2K^−/− MEFs treated with 500 μM H₂O₂ for increasing periods (5 min–6 h). Untreated WT MEFs were used as a negative control and WT MEFs treated with 500 μM H₂O₂ for 5 min were used as a positive control.

(B) Wild-type and eEF2K^−/− MEFs were exposed to 500 μM H₂O₂ for increasing periods (5 min–4 h), and eIF2α phosphorylation was assessed by immunoblotting.

(C) Quantification of eIF2α phosphorylation. The graph represents the ratio of phosphorylated to total eIF2α quantified from the western blots in Figure 6B. The intensities were quantified using Licor Image Studio software. The individual symbols represent individual data points. The individual data points represent duplicated individual repeat experiments.

(D) Cells were treated with the indicated doses (0.5–10 mM) of H₂O₂ for 1 h. An MTT assay was performed to assess cell viability; see Figure S2C for verified results. The graph represents the means ± standard deviations of eight replicates.

(E) Ribosome transit times were analyzed in wild-type, eIF2α^S51A, eEF2K^−/−, and eIF2α^S51A/eEF2K^−/− MEFs treated with 500 μM H₂O₂. The graph represents the mean percent increases in 1/2 transit times ± standard deviations of three independent experiments each done in technical triplicates. The individual symbols represent summaries of the individual experiments. *p value between WT and eIF2α^S51A = 0.017, p value between WT and eEF2K^−/− = 0.044, p value between eIF2α^S51A and eEF2K^−/− = 0.024. Statistical significance was determined by t test.

(F) The graph represents the normalized mean fold change of 1/2 transit times ± standard deviations of the same experiments as shown in Figure 6D. The individual symbols represent summaries of the individual experiments. *p value WT = 0.313, p value eIF2α^S51A = 0.007, p value eEF2K^−/− = 0.108, p value eIF2α^S51A/eEF2K^−/− = 0.314. Statistical significance was determined by t test.

(G) Cells were treated with 1 mM H₂O₂ or 50 μg/mL CHX or untreated. After 15 min, [³⁵S]-methionine was added for an additional 45 min. Protein synthesis levels were then quantified and adjusted according to the total amount of protein. The graph represents normalized means ± standard deviations of at least two independent experiments each done in technical triplicates. The individual symbols represent summaries of the individual experiments. *p value eEF2K^−/− = 0.004, p value eIF2α^S51A/eEF2K^−/− = 0.0007. Statistical significance was determined by t test.

(H) Cells were treated with 1 mM H₂O₂ for increasing times indicated and labeled with [³⁵S]-methionine for 5 min. Protein synthesis levels were then quantified and adjusted according to the total amount of protein. The graph represents the normalized means ± standard deviations of at least two independent experiments each done in technical triplicates.