Figure 3.

eIF5B represses ATF4 translation by a uORF-dependent mechanism. (A) Schematic representation of the ATF4-firefly luciferase reporter fusions used in this study, all transcribed from a minimal TK promoter. Note that Mut1 and Mut2 possess a start codon mutation (ATG to AGG) that inactivates the regulatory functions of uORF1 and uORF2, respectively. Mut1+2 possesses both mutations. (B) Control or eIF5B-depleted HEK293T cells were transfected with the above-mentioned plasmids (48 h post siRNA transfection). After another 48 h, the luminescence from firefly luciferase was measured and normalized to the steady-state levels of firefly luciferase mRNA (measured by RT-qPCR). (C) Luciferase levels were measured as in panel (B), except that the cells were treated with 5 µg/mL tunicamycin (TUN) for the last 6 h of the incubation prior to harvesting. Data are expressed as mean ± SEM for 3 independent replicates. ** p < 0.01; *** p < 0.001. (D) Immunoblots probing for eIF5B, ATF4, eIF2α, P-eIF2α, or β-Actin (internal control). Numbers above the blots represent their quantitation relative to β-Actin.