(A) Western blot of K562 ISR reporter cell extracts after treatment with thapsigargin (tg) or dTag13 for 3 hr as indicated. (B–D) Förster resonance energy transfer signal as monitored by flow cytometry after 3 hr treatment with (B) dTag13 (EC50 = 5.1 nM; s.e.m. = 0.2 nM), (C) ISRIB ± 83 nM dTag13 (EC50 = 80 nM; s.e.m. = 10 nM), and (D) various stressors (83 nM dTag13, 50 nM tg, ± 1.6 μM ISRIB). The ratio of mScarlet-i/mNeonGreen emission is presented. (E) Western blot of K562 ISR reporter cell extracts treated for 3 hr with ISRIB, tg, and/or dTag13 as indicated. All lanes across gels were loaded with equal total protein. For (A), eIF2Bδ, eIF2Bα, and GAPDH blots, and the ATF4 and eIF2α blots are from the same gels, respectively; the eIF2α-P blot is from its own gel. For (E), eIF2Bδ, eIF2Bβ, and GAPDH blots, ATF4 and eIF2α blots, and eIF2Bα and eIF2α-P blots are from the same gels, respectively. For (B–D), biological replicates: n = 3. All error bars represent s.e.m.