UPF1-NKR mutant is not functional in NMD. HEK cells stably transfected with pKK plasmids were induced for 96 h with doxycycline to simultaneously express an RNAi cassette against UPF1 (UPF1 KD) or luciferase (CTRL KD), and RNAi-resistant UPF1 versions (WT, DE, or NKR) or MBP as a control. (A) The efficiency of the UPF1 KD and the expression levels of the recombinant proteins were assessed by western blotting with the indicated antibodies. Total protein stain served as loading control. (M) Molecular weight marker. (B) Relative mRNA levels of hnRNPL_Protein, hnRNPL_NMD, RP9P, and TRA2B_NMD, normalized to actin mRNA levels, were determined by RT-qPCR. Averages and standard deviations from three independent experiments are shown. Statistical significance was determined by two-way ANOVA and Dunnett's multiple comparisons test against the CTRL KD + MBP condition. Asterisks are only displayed for the comparisons with a P-value smaller than 0.05. (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001, (****) P ≤ 0.0001.
