1. Identification of de novo synthesized proteins in response to ER stress.
Identification of de novo synthesized proteins in response to ER stress.A, HeLa cells were transfected with siRNA against UPF1 or a control siRNA. Control cells were treated with 1.5 mm DTT for 6 h. Protein expression of UPF1 and the ER stress marker BiP was visualized by Western blotting. BiP is up-regulated by DTT treatment but not in UPF1-depleted cells. B–E, HeLa cells stably expressing the NMD reporter Renilla-HBB NS39 treated with 1.5 mm DTT for 6 h. During the treatment cells were also incubated in methionine, arginine and lysine-free DMEM containing AHA and isotope-labeled arginine and lysine. Isotope labels were reversed in biological duplicates. B, Up-regulation of the NMD reporter Renilla-HBB NS39 was measured by luciferase assay. C, RT-qPCR was used to measure the expression level of endogenous NMD target RNAs. D, 5199 proteins were quantified in both biological replicates. Significantly regulated proteins (FDR < 0.05) are highlighted in red. Proteins shown in gray were removed from statistical analysis because of their high standard deviation. R = Pearson correlation coefficient. E, Distribution of the average change of expression after DTT treatment.
Jana Sieber et al. Mol Cell Proteomics 2016;15:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.