Fig. 4
CircPDE5A encodes a novel protein, PDE5A-500aa. A Structure of circPDE5A and predicted amino acid sequence of PDE5A-500aa. The upper panel presents a schematic representation of the predicted IRES and ORF sequences in circPDE5A. The lower panel illustrates the predicted amino acid sequence of PDE5A-500aa. B Detection of initiator protein translation activity of IRES in circPDE5A. The left panel displays wild-type, truncated, or mutated sequences of IRES in circPDE5A cloned between Rluc and Luc reporter genes with independent initiation codons and termination codons. The right panel depicts the examination of relative luciferase activity of Luc/Rluc in the aforementioned vectors. C Three flag-tagged expression vectors were constructed: p-circPDE5A-flag, p-circPDE5A-flag-Mut, and p-PDE5A-500aa to demonstrate the protein-coding function of circPDE5A. D The anti-PDE5A antibody (#22,624–1-AP; Proteintech, Wuhan, China) recognizes both PDE5A and PDE5A-500aa. E WB for the protein levels of PDE5A, PDE5A-500aa, and flag-tagged PDE5A-500aa in KYSE30 cells treated with various vectors. F WB shows the levels of PDE5A and PDE5A-500aa in HEEC cells treated with sh-NC, sh-circPDE5A-1, and sh-circPDE5A-2. G The left panel shows IP of anti-flag antibodies in 293 T cells transfected with circPDE5A-flag, and SDS-PAGE separated the pull-down protein. Silver-stained protein bands near 58 kDa are manually excised and analyzed by LC–MS/MS to identify the peptide sequence. The right panel shows the LC–MS/MS identification of the unique peptide sequence of PDE5A-500aa (RNGQCMVC). H Co-IP indicates that circPDE5A-flag encodes PDE5A-500aa-flag. I Subcellular localization of PDE5A-500aa in KYSE30 and KYSE450 cells detected by immunofluorescence. Bar represents 5 μm. J WB to detect the protein levels of PDE5A and PDE5A-500aa in normal esophageal epithelial cells and ESCC cells. K WB to detect PDE5A-500aa level in ESCC and adjacent normal tissues. L Kaplan–Meier analysis of the correlation between PDE5A-500aa level and OS in patients with ESCC. ***P < 0.005