The effects of salubrinal on the ISR pathway and protein translation in human primary erythroid progenitors. (A) Western blot analysis reveals p-eIF2α is increased with salubrinal (Sal) treatment on day 15 of the culture. In contrast, ATF4 and GADD34 protein are not increased with Sal treatment relative to an untreated control (-). Numbers below each lane denote the ratio of p-eIF2α/eIF2α quantified by ImageJ software. Total eIF2α and GAPDH are used as loading controls. (B) The mRNA expression of ATF3 and CHOP are not increased at 1 to 6 hours after Sal treatment on day 15. Expression is reported as fold change relative to untreated negative control. Error bars represent ± standard error of the mean of 3 independent experiments (2 unique donors). (C) ATF3 and CHOP mRNA levels are not enhanced with Sal treatment over the course of differentiation. Expression is reported as fold change relative to the untreated control (Untx) on day 7. Sal (3 μM and 6 μM) were treated on days 15 and 18. Error bars represent ± standard error of the mean of 3 independent experiments (2 different donors). (D) Representative polysome profile on day 18 after 6 hours of Sal treatment reveals a polysome to monosome shift, indicative of halted translation initiation and reduced translation.