Figure 1.

GCN2 amino acid stress signaling genetic reporters.A, pathway immunoblotting analysis of the mammalian GCN2-ISR. 3T3 WT and GCN2-deficient (Eif2ak4^−/−) cells were starved in Leu-deficient media (Leu starvation) for the times indicated (h). Lysates were probed for activation of GCN2 (p-GCN2 T898), total GCN2, ATF4 as a marker of the ISR. GRB2 was used as a loading control. B, kinetics and GCN2 dependency of CHOP expression in response to leucine starvation. Data are representative of a minimum of three independent experiments. C, schematic representation of the Ddit3 locus (encoding CHOP) with the position of the sgRNAs (guides). Homologous repair plasmids are indicated. D, representative live-cell imaging showing the mCherry signal following Leu starvation and the dependence on GCN2. sgRNA, single-guide RNA.