Fig. 1. ISRIB reverses attenuated translation and accelerates eIF2B GEF activity towards eIF2(αP) in vitro.

(A) Immunoblot of newly-synthesized puromycinylated proteins in extracts of untreated CHO cells or cells exposed to the ISR-inducing agent thapsigargin (Tg 300 nM, 30 minutes) in the presence or absence of trans ISRIB (100 nM). Phosphorylated (P-eIF2α) and total eIF2α were detected in the immunoblots below. Quantified signal intensities are shown in Fig. S5. (B) Dose-response of ISRIB-stimulation of translation in reticulocyte lysate fitted to a non-linear trace. Shown are mean ± SEM (n = 3) and EC₅₀ (for active trans-ISRIB). Note the inactivity of cis-ISRIB. (C) GEF activity as reflected in time dependent decrease in fluorescence of weakly and heavily phosphorylated eIF2 loaded with Bodipy-FL-GDP and incubated with unlabeled GDP in the presence or absence of cell lysate (μg). Shown is a mean of three independent measurements. (D) Relation between the initial velocities of the release of Bodipy-FL-GDP from heavily phosphorylated eIF2 and ISRIB concentration, fitted to a non-linear trace. Shown are mean ± SEM (n = 3) and EC₅₀ for trans-ISRIB. (E) GEF activity reflected in the initial velocities of GDP release reactions with CHO cell lysate (samples 1-4), wildtype or mutant eIF2α^S51A/S51A mouse embryonic fibroblast lysate (MEFs, samples 5-8) and Bodipy-FL-GDP loaded eIF2 of the indicated eIF2α genotype. Shown are mean ± SEM (n = 3 for samples 1-4 and n = 6 for samples 5-8). *P < 0.05, **P < 0.01 (Student’s t test). (F) As in “E”, but with purified eIF2B and Bodipy-FL-GDP loaded non-phosphorylated and phosphorylated eIF2. Shown are mean ± SEM (n = 8). *P = 0.012, **P = 0.0054 (Student’s t test). (G) Coomassie-stained SDS-PAGE of the purified eIF2B used in “F”. The five subunits of eIF2B and PRMT5 (*, a non-specific contaminant) are noted.