Fig. 6

circZKSaa prevents AKT activation through mTOR. HCC-LM3 and Hep3B cells were transfected corresponding vectors (circZKSCAN1-mut\circZKSCAN1\sh-scramble\sh-circZKSCAN1\Vector\circZKSaa) to detect the proliferation signal pathway. A The expression level of β-catenin, ERK, phosphorylated ERK, AKT, T308 site phosphorylated-AKT and S473 site phosphorylated-AKT were determined via Western blot, and GAPDH expression level served to indicate the amount of loading proteins. B The protein expression level of PDK1, phosphorylated PDK1, mTOR and phosphorylated mTOR were determined via Western blot, and GAPDH expression level served to indicate the amount of loading proteins. C After Co-IP by HA antibody and separation by SDS-PAGE, the protein sample which stained by silver staining was analyzed by mass spectrometry. D Co-IP analyses by HA and mTOR antibody determined the interaction between circZKSaa and mTOR. E Immunofluorescence staining of mTOR and circZKSaa-HA in HCC-LM3 cells, Scale bars = 20 μm. F Western blot assay were used to detect MDM2, phosphorylated-MDM2, S6K1 and phosphorylated-S6K1 in both HCC-LM3 and Hep3B cells transfected with vectors (circZKSCAN1-mut\circZKSCAN1\sh-scramble\sh-circZKSCAN1\Vector\circZKSaa), GAPDH expression level indicated the amount of loading proteins. Quantitative data from three independent experiments are presented as mean ± SEM (error bars). T test was performed for significant analysis, significant differences are indicated with * for p < 0.05, ** for p < 0.01 and *** for p < 0.001