Multiple unexpected TPI splice isoforms derived from the TPI WT plasmid. (A) Schematic drawing of the used TPI WT reporter plasmid expressed under the control of a CMV promotor. The numbers indicate the exon numbers and the ATG and TC indicate the start and stop of the ORF. The four HBB repeats can be used as probe-binding sites for northern blot analysis. (B) RT-PCR results of untransfected HeLa cells (lanes 1 and 2) and HeLa cells transiently transfected with 1 µg of the pCI-TPI-WT plasmid for 24 h (lanes 3 and 4) that were either untreated (lanes 1 and 3) or treated with 50 µg/mL CHX for 6 h (lanes 2 and 4). The genes of interest were amplified with specific primer pairs (cTPI + pTPI: #5664/#5665, 26 cycles, pTPI: #4324/#5665, 18 cycles, HGH transfection control #1224/#1225 26 cycles, NMD control SRSF3 #4003/#4004 35 cycles), separated on a 10% PAA gel and visualized by EtBr staining and UV light exposure. The TPI bands were excised, reamplified, and sequenced. Numbers in brackets represent the exon numbers that were included in the splice isoform. Predicted NMD targets with frameshift mutations are highlighted in red letters, while green letters represent presumable not-NMD targets with intact ORFs.
