tRNA synthetase inhibition induces dose-dependent eIF2α phosphorylation similar to canonical stressors. Western blot of P-eIF2α and total eIF2α from U-2 OS cells treated with arsenite (As) at 25–250 μM for 1 h (A) or thapsigargin (Tg) at 0.04–1 μM for 1 h (B). (C) P-eIF2α and total eIF2α western blot with nascent (BONCAT) and total protein from U-2 OS cells treated with DMSO (0.2%) control, arsenite (500 μM, 30 min), or halofuginone (HF) at 0.02–20 μM for 4 h. No copper controls are shown at left. (D) Western blot of U-2 OS cells treated with borrelidin (borr; 1–125 μM for 4 h) or ethanol (EtOH) carrier. Representative western blots with total protein loading controls and molecular weights in kDa shown at left with quantification of n = 3 independent replicates with averages ± SEM and replicate values shown as green, gray, and pink points at right. Significance assessed with ordinary one-way ANOVA and Tukey's multiple comparisons tests with (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.
