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Figure 5 Alternative splicing analysis using minigenes by transfection experiment in P19 cells
(A, C, E) Schematic representation of 5-HT3R-A, Actn1 and CUGBP2 minigenes. The mouse genomic fragments for minigenes were inserted between CMV promoter and SV40 poly(A) site in a mammalian expression vector. The black arrows indicate the positions of primers used for amplification of alternatively spliced products, and the grey arrow indicates the position of T7 primer for cDNA synthesis. Five micrograms of each minigene containing expression vector were transiently transfected in P19 cells and were cultured in different treatment conditions for 24 h. (B) The transfected cells were cultured in the absence or presence of RA treatment. The percentage of inclusion of 5-HT3R-AL was determined by densitometry. (D) In the case of Actn1, the cells were cultured in the absence or presence of aggregation. The NM and SM exon inclusions were determined by densitometry. (F) For CUGBP2, the cells were treated in the absence or presence of RA treatment, and the absence or presence of cell aggregation. By using densitometry, the percentage change of γ and δ isoforms was determined. In each case, the percentage of each splice product was calculated over the total of spliced products. All the experiments were repeated more than three times, and similar results were obtained. P-values were determined by Student’s t testing (*P<0.05). The asterisk values were determined compared with no treatment. The upper bold boxes indicate values of relative ratios of the isoforms.