Figure 4. GADD34 mediates eIF2α dephosphorylation and controls translation in DCs.

WT and GADD34ΔC BMDCs were stimulated with LPS (100 ng/ml) for the indicated times. (A) Levels of p-eIF2α, total eIF2α, p-eIF2β, total eIF2β, p-eEF2, total eEF2, P-S6, and total S6 were detected by immunoblot (top left) and quantification is shown in the different panels. (B) The speed of translation elongation was measured by SunRISE after 3 h of incubation with LPS. Harringtonine (2 μg/ml) was added at different times up to 90 s prior incubation with puromycin during 10 min. Flow intracellular staining was performed in cDC2 using an α-puromycin antibody. The total decay of puromycin mean fluorescence intensity between WT and GADD34ΔC in steady-state and upon activation indicates that translation initiation and elongation speed is decreased in GADD34-deficient DCs. Flt3-L BMDCs were pretreated with 2 μM of the TBK1 inhibitor (MRT67307) for 1 h, before stimulation with LPS (100 ng/ml) for indicated times. (C) mRNA levels of GADD34 were measured by qRT-PCR and normalized to the housekeeping gene (GAPDH) level. (D) Immunoblot detection of p-eIF2α, total eIF2α, P-IRF3, and total IRF3. Quantification is represented on the right. (A, B, C) Statistical analysis was performed using the Wilcoxon test (A, C, B), and Mann–Whitney test (*P < 0.05 and ****P < 0.0001). (D) All data are representative of n = 3 independent experiments except (D), n = 2.