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. 1998 Aug 18;95(17):10009–10014. doi: 10.1073/pnas.95.17.10009

Figure 3.

Figure 3

Transient expression of R844C hUPF1 cDNA in COS cells abrogates the nonsense-mediated decrease in the abundance of GPx1 mRNA. COS cells were transiently transfected with a test plasmid pmCMV-GPx (harboring either a TGC cysteine codon or a TAA nonsense codon at position 46), the reference plasmid phCMV-MUP, and pCI-neo-hUPF1 (harboring either Wt or R844C hUPF cDNA). The amounts of each plasmid used were, respectively, 10 μg, 3 μg, and 37 μg or 10 μg, 3 μg, and 25 μg. An appropriate amount of a fourth plasmid was added to each transfection to bring the total amount of introduced DNA to 50 μg. Nuclear and cytoplasmic RNA was purified, and the amounts of GPx1 and MUP mRNAs were quantitated by using RT-PCR. GPx1 and MUP mRNAs generate 420-bp and 199-bp products, respectively. The level of mRNA from each mCMV-GPx1 allele was normalized to the level of MUP mRNA. Normalized values for GPx1 mRNA harboring UAA(46) then were calculated as a percentage of the normalized value of GPx1 mRNA harboring UGC(46) in the presence of either the Wt hUPF1 gene or the R844C hUPF1 gene, each of which was considered as 100. Percentages differed between two independently performed experiments by no more than 9%.