Fig. 4

CircEIF4G3 acts as a scaffold to promote δ-catenin ubiquitin degradation by TRIM25. (A) The expression of δ-catenin protein in GC cells with or without circEIF4G3 overexpression after treatment with MG-132 (40 μM) for 6 h was determined by western blot. (B) Protein biosynthesis in GC cells was blocked with cycloheximide (CHX). The protein levels of δ-catenin in GC cells of indicated groups were determined at indicated time points by western blot. The corresponding quantification curve was exhibited. (C) GC cells were transfected with ubiquitin (Ub) and circEIF4G3 overexpressing plasmid and treated with MG-132. The ubiquitination of δ-catenin was determined by immunoprecipitation (IP) with δ-catenin antibody followed by Western blot with ubiquitin antibody. (D) The binding of TRIM25 and δ-catenin in GC cells was detected by Co-IP. (E) Immunofluorescent staining for the co-localization of δ-catenin protein (green) and TRIM25 protein (red) in GC cells. Scale bar = 25 μm. (F) δ-catenin protein levels in control and TRIM25 overexpressing GC cells were examined by western blot. (G) The ubiquitination of δ-catenin in GC cells with TRIM25 overexpression. (H) GC cells were transfected with circEIF4G3 overexpressing plasmid and the binding of TRIM25 to δ-catenin was determined by Co-IP and western blot