Fig. 2. NMD of cytoplasmic GPx1 mRNA is not restricted to newly exported mRNA. HeLa Tet-Off cells were transiently transfected with two plasmids: pTRE-GPx1(TGC) or pTRE-GPx1(TAA) test plasmid, in which the GPx1 gene harbors either a cysteine codon or a nonsense codon in place of the Sec codon and is under the control of the doxycycline-repressible TRE promoter, and the phCMV-MUP reference plasmid. After 24 h, doxycycline was added to shut off GPx1 gene transcription, and nuclear (A) and cytoplasmic (B) RNA was harvested at the specified times. Lanes labeled ‘+’ indicate that doxycycline was present throughout the experiment and provide a measurement of the amount of leaky transcription. Quantitations of nuclear GPx1 pre-mRNA demonstrate that the shut-off in transcription was evident by 2.5 h after doxycycline addition. For each lane, the level of GPx1 mRNA was normalized to the level of MUP mRNA and is presented as a percentage of the normalized level of either UGC- or UAA-containing mRNA, which was defined as 100. Half-lives are calculated to be 8–10 h for GPx1(UGC) mRNA and 2.5 h for GPx1(UAA) mRNA (C).