Regulation of CReP turnover and impact on eIF2α phosphorylation.

<p>(A) CReP is depleted upon DNA damage but not proteostatic stress. Cells were treated with 1 μM thapsigargin, 3 μg/mL camptothecin, or 300 J/m² UV for the indicated time; all samples not treated with UV were mock-treated and all samples were given the same total volume of the solvent DMSO. (B) CReP turnover upon DNA damage depends at least in part on βTRCP. Cells were transfected for 48 hours with an empty vector or shRNA targeting βTRCP1 and 2, then irradiated with 300 J/m² UV-C. CReP levels are quantitated below, and the half-life calculated from the linear (0–2 hr.) part of the timecourse. (C) CReP depletion and full eIF2α phosphorylation in UV depends on CRLs. Cells were treated with UV with or without MLN4924 for the times indicated. (D) CReP depletion in primary human fibroblasts depends on CRLs. Primary human fibroblasts were treated with 1 μg/mL camptothecin for 6 hours, with 1 μM MLN4924 where indicated. (E) The 31A allele of CReP is stable even upon treatment with DNA damage and cycloheximide. Cells were transfected with wildtype or mutant CReP, then pre-treated for 2 hours with 3 μg/mL camptothecin before addition of cycloheximide. (F) Expression of a stable allele of CReP prevents phosphorylation of eIF2α in response to UV treatment. Cells were transfected with tagged wild type or mutant CReP, then treated with UV for the indicated times.