Combinatorial adjustments mitigate alternatively spliced transcripts. (A) Schematic drawing of the used TPI reporter construct. The 3′ end of exon 3 and the 5′ end of intron 3 were enlarged to show the introduced mutation and their effect on the HEXplorer plot. The SD was modified by a single point mutation (gAGGTtAGTAg > gAGGTAAGTAg, HBS 16.9 > 20.8). (B) RT-PCR results were obtained from the transfection of HeLa cells with the modified plasmids. HeLa cells were transiently transfected with the depicted reporter constructs for 24 h. Six hours before the total RNA was harvested, the cells were treated with 50 µg/mL CHX. The RNA was reverse transcribed and amplified with specific primer pairs (#4324/#5665 pTPI, 18 cycles). HGH (#1224/1225, 26 cycles) served as transfection control and SRSF3 (#4003/#4004, 35 cycles) as control for CHX treatment. The PCR products were separated on a 10% PAA gel, visualized by EtBr staining and UV light exposure. (C) Schematic drawing of the used TPI reporter construct. The 3′ end of exon 2 and the 3′ end of intron 2 were enlarged to illustrate the inserted mutations. The HEXplorer profile was modified by two silent point mutations within exon 2 (ACT-GGG-GAG-ATC-AG > ACC-GGA-GAG-ATC-AG), and two point mutations within intron 2 (CAG/GTGAGATCGAGGTGG > CAG/GTGAGATCTAGGCGG). (D) HeLa cells were transiently transfected with one of the depicted reporter constructs for 24 h. Six hours before the total RNA was harvested, the cells were treated with 50 µg/mL CHX. The RNA was reverse transcribed and amplified with specific primer pairs (#4324/#5665 pTPI, 20 cycles). HGH (#1224/1225, 26 cycles) served as transfection control and SRSF3 (#4003/#4004, 35 cycles) as control for CHX treatment. The PCR products were separated on a 10% PAA gel, stained with ethidium bromide, and visualized via UV light.
