1. Identification of de novo synthesized UPF1 target proteins.
Identification of de novo synthesized UPF1 target proteins. HeLa cells stably expressing the NMD reporter Renilla-HBB NS39 were transfected with UPF1 siRNA or a control siRNA. Two days after transfection DMEM was replaced with methionine, arginine and lysine-free DMEM containing AHA and isotope-labeled arginine and lysine. Isotope labels were reversed in biological duplicates. Cells were harvested after 6 h of incubation. NMD inhibition was controlled by expression analysis of the NMD reporter and known endogenous NMD targets. The remaining cell lysate was used for enrichment of de novo synthesized proteins and subsequent analysis by LC-MS/MS. A, Luciferase assay reveals a twofold up-regulation of Renilla-HBB NS39 on protein level upon UPF1 depletion. B, RT-qPCR analysis of endogenous NMD target RNAs. RPL13A serves a non-NMD target control. UPF1 mRNA was downregulated to 40% by siUPF1. C, 4898 proteins were identified and quantified in two biological replicates. Proteins that were regulated with statistical significance (FDR < 0.05) are shown in red. UPF1 is the most downregulated protein (down to 27%). R = Pearson correlation coefficient. D, Distribution of the average change of expression after UPF1 depletion.
Jana Sieber et al. Mol Cell Proteomics 2016;15:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.