Characterization of oligo#5 and its derivatives Characterization of oligo#5 and its derivatives Titration of oligo#5. An increasing amount of oligo#5 was added to HEK293 cells stably expressing the serotonin 2C receptor reporter minigene, and the RNA was analyzed by RT–PCR. –RT: negative control without reverse transcriptase.Quantification of three independent experiments. The half‐maximal effect was seen at 13.1 nM concentration. Error bars indicate standard deviation.Influence of oligo#5 shortening on alternative splicing of the serotonin 2C receptor. Oligo#5: 5′ AGU AUU GAG CAU AGC CGC 3′ (18mer); Oligo#5‐3: 5′ GAG CAU AGC CGC 3′ (12mer); and Oligo#5‐10: 5′ G CAU AGC CGC 3′ (10mer) were used in transfection assays analyzed by RT–PCR.Statistical analysis of the effect of oligonucleotide shortening. The effect of all oligos testes is significant, oligo#5: ***P = 0.0015; oligo#5‐3: *P = 0.031; and oligo#5‐10: *P = 0.048 (Student's t‐test), n = 6. Error bars indicate standard deviation.Influence of cy3 label on the 5′ and 3′ end of oligo#5. Using a phosphate linker, Cy3 was added to the 5′ or 3′ phosphate, respectively, and the oligonucleotides were assayed by RT–PCR. Oligo#5 with cy3 at the 5′ end showed 86% inclusion, with Cy3 at the 3′ end 17% exon inclusion.Statistical analysis of the effect of oligonucleotide modification. The effect is significant, **P = 0.009 (Student's t‐test), n = 4. Error bars indicate standard deviation. Source data are available online for this figure. Zhaiyi Zhang et al. EMBO Mol Med. 2016;8:878-894 © as stated in the article, figure or figure legend