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. Author manuscript; available in PMC: 2018 Sep 30.
Published in final edited form as: Science. 2018 Mar 30;359(6383):1533–1536. doi: 10.1126/science.aar5129

Figure 3. Sensitivity to ISRIB analogs selects for a divergent palette of mutations in codon 188 of Eif2b2.

Figure 3

(A) Histograms of the ISR-responsive CHOP::GFP fluorescent reporter activity, induced by histidinol (HIS+, 0.5 mM) in ISRIBRES, ISRIBSEN, compound 075BSEN and compound 084SEN sub-pools, selected for their responsiveness to ISRIB or its analogs (2.5 µM) from a population of originally ISRIBRES Eif2b2H188X mutant cells.

(B) Bar graph of the distribution of residues identified at Eif2b2 codon 188 in phenotypically divergent pools of CHO-K1 cells. The number of sequenced reads in ISRIBRES (plum), ISRIBSEN (orange), compound 075BSEN (blue) and compound 084SEN (cyan) pools encoding each amino acid (* - stop codon, X – ambiguous) is plotted.

(C) Plot of the relative FP signal arising from samples with fluorescein-labeled AAA2-101 (2.5 nM) bound to purified hamster eIF2B (30 nM) in the presence of the indicated concentration of unlabeled ISRIB introduced as a competitor (represented on a log10 scale). Shown is a representative of two independent experiments. The fitting curve and EC50 was generated using “agonist vs. response” function on GraphPad Prism.

(D) A plot of the FP signal arising from fluorescein-labeled AAA2-101 (2.5 nM) as a function of the concentration of wildtype (wt) or mutant eIF2B (δL180F or βH188K) in the sample. Shown are mean ± SD (n=3). Concentrations of eIF2B are represented on a log10 scale.