Fig. 3. The 5-HT6R overexpression attenuates the splicing activity of Nova-1 centered on the excision of GnRH intron A. (A) The structure of the GnRH gene and positions of the primers (top). A standard curve was constructed to analyze the quantitative ratio of 1234 and 1A234 cDNAs by competitive PCR using 10 pg of the 1A234 and serial dilutions of the 1234 gene (bottom). (B) Nova-1 increased the rate of intron A excision of GnRH gene in dose-dependent manner when GnRH minigene and Nova-1 were transiently transfected into NIH3T3 cells (F(3, 8)=29.79, p=0.0001; Dunnett's post hoc test: ***p<0.001). (C) 5-HT6R decreased the splicing activity of Nova-1 in dose-dependent manner (F(4,10)=18.92, p=0.0001; Dunnett's post hoc test: **p<0.01, ***p<0.001). (D) C-terminal truncated 5-HT6R (5-HT6RΔCT) did not affect the splicing activity of Nova-1 (F(3, 8)=0.4302, p=0.7371). (E) 5-HT6R dose-dependently decreased the splicing activity of Nova-1 in GT1-1 neuronal cell line in which GnRH and Nova-1 are endogenously expressed (F(4, 10)=12.69, p=0.0006; Dunnett's post hoc test: **p<0.01). (F) The splicing activity of Nova-1was not affected by 5-HT6RΔCT in GT1-1 neuronal cell line (F(4. 5)=0.194, p=0.9314). Data are expressed as means±S.E.M. (n=3 per group).
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