Fig. 4: ISRIB impairs the degradation of DRiPs.

a Effect of ISRIB on DRiP synthesis. Analysis of puromycin incorporation in MelJuSo cells pre-treated without (-ISRIB) or with ISRIB (+ISRIB). Cells were left untreated (- HS) or exposed to 43 °C for 30 min (+HS). b Effect of ISRIB on localization of DRiPs. Representative micrographs of MelJuSo cells that were pre-treated with (+ISRIB) or without ISRIB (-ISRIB). Cells were exposed to 43 °C for 30 min (+HS) in the presence of puromycin. Puromycin and the stress granule marker TIA1 were visualized by immunostaining. Images were captured with a laser scanning confocal microscope. Scare bar, 10 μm. c Western blot analysis of ubiquitylated DRiPs. MelJuSo cells expressing Ub-YFP that were incubated in the absence of presence of ISRIB for 30 min before left untreated or subjected to 43 °C heat shock for 30 min with or without 5 ug/ml puromycin. The cells were lysed as indicated and ubiquitylated proteins were pulled down with tandem ubiquitin binding entities (TUBEs). The input and pulldown samples were analyzed by immunoblotting with antibodies directed against ubiquitin and puromycin. d Western blot analysis of Triton X-100 solubility assay of MelJuSo cells incubated with or without ISRIB for 30 min before left untreated (−HS) or subjected to 43 °C heat shock for 30 min (+HS). Ubiquitylated proteins were detected in total lysates (I), insoluble pellet fraction (P), and soluble fraction (S). GAPDH is used as a control for soluble proteins.