The UPF1-NKR mutant exerts a dominant-negative effect. (A,B) HEK cells were transiently transfected to express UPF1-GFP (WT, DE, NKR, and DE.NKR variants) or GFP as a control. (A) Relative mRNA levels of hnRNPL_Protein, hnRNPL_NMD, RP9P, and TRA2B_NMD, normalized to 28S rRNA and actin mRNA levels, were determined by RT-qPCR 48 h after transfection. Averages and standard deviations from four independent experiments are shown. (B) The expression level of the overexpressed recombinant proteins was assessed by western blotting using the indicated antibodies. Tubulin served as loading control. (M) Molecular weight marker. (C,D) HEK cells stably transfected with pKK plasmids were induced for 48 h with doxycycline to express UPF1-3xFLAG proteins (WT, DE, NKR, or DE.NKR) or MBP-3xFLAG as a control. (C) Relative mRNA levels were determined as in A. Averages and standard deviations from five independent experiments are shown. (D) Western blotting assessing the expression of the overexpressed recombinant proteins as in B, using the indicated antibodies. GAPDH served as loading control. Statistical significance in RT-qPCR data was determined by two-way ANOVA and Dunnett's multiple comparisons test against the GFP (A) or MBP (C) controls. 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.
