Fig. 3
CircCAPG encodes a novel protein named CAPG-171aa. (A) An open reading frame (ORF) of circCAPG and its start and end codon were labeled (Left). All exons and ORF of CAPG and its start and end codon were labeled (Right). Red marked CAPG-171aa specific amino acid sequence. Blue marked the same coding sequence of CAPG and circCAPG. (B) Three modified dual-luciferase vectors, luciferase-fired, EMCV-IRES-inserted, and circCAPG-IRES-inserted vectors were established to determine IRES activity of circCAPG. Luciferase-fired and EMCV-inserted vectors work as negative and positive controls, respectively. (C) Mutant IRES (circCAPG-FLAG-IRES-mut) and ATG (circCAPG-FLAG-ATG-mut) sequence of circCAPG was designed to validate the coding potential of circCAPG. qPCR of circCAPG and linear CAPG was then tested. (D) IB of CAPG-171aa in circCAPG-FLAG and circCAPG-FLAG-IRES-mut cells. (E) IB of CAPG-171aa in circCAPG-FLAG and circCAPG-FLAG-ATG-mut transfected 293T. (F) IB of CAPG-171aa in circCAPG-FLAG through Immunoprecipitating (IP) FLAG antibody. The right graph shows the amino acid sequences of the junction site of circCAPG determined by mass spectrometry (MS). All error bars represent SEM, two-tailed Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ns denotes no significance