Fig. 4

Verification of the coding ability of circMAPK1. a Upper panel, the putative open reading frame (ORF) in circMAPK1. Lower panel, the sequences of putative ORF are shown. b Left panel, the wild or mutate type of IRES was cloned between the Rluc and Luc reporter genes with independent start (AUG) and stop (UGA) codons. Right panel, the relative luciferase activity was tested. c Illustration of MAPK1 sequence and MAPK1–109aa sequence. The antibody used in the study recognized both proteins. d The total protein of HEK293T cells transfected with circMAPK1 and IRES-mut plasmids was separated by SDS-PAGE. Immunoblotting confirmed the overexpression of MAPK1–109aa. Cut the difference gel band between 10 kDa and 15 kDa and perform LC-MS/MS analysis. The identified MAPK1–109aa amino acids are shown in red. e MAPK1 and MAPK1–109aa expression were detected in GC tissues and its paired normal tissues. f MAPK1 and MAPK1–109aa expression were detected in cultured GC cell lines. g Semi-quantitative analysis of MAPK1–109aa in GC tissues. h Overall survival analysis based on MAPK1–109aa expression in 40 GC patients. i The expression of MAPK1 and MAPK1–109aa was detected in MKN45 cells transfected with empty vector, IRES mutated circMAPK1 vector, circMAPK1 vector and MAPK1–109aa vector. j The expression of MAPK1 and MAPK1–109aa was detected in GES-1 cells transfected with control shRNA or circMAPK1 shRNAs. k Flag tagged MAPK1–109aa was transfected into MKN45 cells. Immunofluorescence staining using anti-Flag was performed to show the MAPK1–109aa cellular localization. Scale bars: 20 μM. Graph represents mean ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001