qRT-PCR analysis of relative exon usage. (A) Schematic drawing of the used TPI reporter constructs and primer spacing for qRT-PCR analysis. The numbers indicate the exon numbers, and the ATG and TC indicate the start and stop of the ORF. To distinguish between cellular and plasmid TPI, a forward primer specific for the plasmid promotor region was used (#4324). For amplification of the constitutive transcript containing exons 2 and 3, an exon junction primer was used (#5736). For amplification of the NMD-sensitive splice variant where exons 2 and 3 are skipped, an exon junction primer (#7188) was employed. (B) Results from qRT-PCR analysis are depicted in the bar graph showing the relative E1-4 splicing compared to E2/3 constitutively spliced full-length transcript variants (%) for the pTPI WT, pTPI WT adjusted, pTPI PTC, and pTPI PTC adjusted constructs. E1-4 splicing and E2/3 splicing were normalized to GAPDH (#3602/3603). Data are shown for both untreated and 50 µg/mL CHX-treated conditions. Error bars represent standard deviations from three independent replicates.
