Fig. 3

CircMAP3K4-455aa is a novel peptide encoded by circMAP3K4. A Schematic illustrations of circMAP3K4 (left panel) with ORF amino acid sequence (right panel). B Schematic illustrations of plasmid constructs for circMAP3K4-ORF fused with GFP or Flag tag. C Immunofluorescence staining showing HEK293T cells transfected with circMAP3K4-ORF or circMAP3K4-ORF-mut. D Western blot analysis of circMAP3K4-455aa expression using anti-MAP3K4, anti-flag, or anti-GFP antibodies. E Western blot analysis of circMAP3K4-455aa expression in circMAP3K4 and circMAP3K4-ORF overexpressed cells. F LC–MS/MS analysis of the MAP3K4 peptide sequence. G Sucrose fractionation assays examining circMAP3K4 distribution with ribosomes with GAPDH as internal control. H Western blot analysis of circMAP3K4-455aa expression in 10 cases of HCC and paired liver tissues. ORF, open reading frame; IRES, internal ribosome entry site; GFP, green fluorescent protein; cMAP3K4, circMAP3K4; cMAP-mut, circMAP3K4-mut; cORF, circMAP3K4-ORF; LC–MS/MS, liquid chromatography with tandem mass spectrometry