The ISR pathway is activated by salubrinal in K562 cells. (A) A simplified diagram of the ISR pathway showing both activation and negative regulatory components. (B) Western blot analysis reveals 3 μM and 6 μM salubrinal (Sal) enhances p-eIF2α at 3 and 6 hours. These results were compared with 1 μM TSG as a positive control. Numbers below each lane denote the ratio of p-eIF2α/eIF2α quantified by ImageJ software. GAPDH and total eIF2α were evaluated as loading controls. (C) 3 μM and 6 μM Sal augment downstream ISR signaling to a similar extent as 1 μM TSG, as shown by western blot analysis of ATF4 and GADD34 relative to GAPDH loading control. (D) Sal increases the mRNA expression of ATF3 and CHOP dose dependently at 3 and 6 hours post-treatment. Transcript levels at each time point are reported as fold increase relative to a dimethylsulfoxide (DMSO) control. Error bars denote ± standard deviation of 3 technical replicates.