Fig. 2

CircAXIN1 encodes a novel protein AXIN1-295aa. a An illustration showing the construction of FLAG-tagged circRNA. b CircAXIN1 potentially encodes a novel protein, AXIN1-295aa. Immunoblotting of lysates from cells transfected with FLAG-tagged circRNAs. c The circRNAs were successfully overexpressed as determined by quantitative real-time PCR using divergent primers. d AXIN1-295aa is encoded by the circularized RNA, circAXIN1. The plasmids with start codon mutations and with deletion of downstream flanking sequences were unable to generate AXIN1-295aa. The linearized circAXIN1 ORF was cloned into a CMV-induced expression vector (linear AXIN1-295aa) to serve as a positive control. e An illustration of the IRESs and ORF of circAXIN1. Both IRESs are active. The IRES was cloned between mCherry and GFP, as shown. The constructs were transfected into 293 T cells and images were taken to show the expression of mCherry and GFP. f CircAXIN1 has two active IRESs. The two IRESs and partial IRESs were constructed between tandem Rluc and Luc reporters with independent start and stop codons. The constructs were transfected into cells. The relative luciferase activity was determined