Fig. 5

AGEs mediate β-catenin phosphorylation and Y142 is the key site of phosphorylation. A HUVECs were treated with AGEs (100 μg/ml) for different times, and then serine/threonine phosphorylation and tyrosine phosphorylation level of β-catenin were detected by western blot. One representative cropped blot of three independent experiments is shown. B Schematic diagram of constructing the three types of β-catenin multi-site mutant (Y to F) plasmids. Vertical line in green means tyrosine (Y) while line in red means the site is mutated to phenylalanine (F). Illustrated by IBS software. C β-catenin multi-site mutant (Y to F) plasmid was transfected into 293T cells followed by AGEs treatment, and then tyrosine phosphorylation level of β-catenin was detected by western blot. n = 4. One representative cropped blot of four independent experiments is shown and the phosphorylation protein level is calculated relative to each group without AGEs treatment, defined as “1”. D Schematic diagram of constructing the four types of β-catenin single-site mutants (Y to F) plasmids. Vertical line in green means tyrosine (Y) while line in red means the site is mutated to phenylalanine (F). Illustrated by IBS software. E The β-catenin single-site mutant (Y to F) plasmid was transfected into 293T cells followed by AGEs treatment, and then tyrosine phosphorylation level of β-catenin was detected by western blot. n = 4. One representative cropped blot of four independent experiments is shown and the phosphorylation protein level is calculated relative to each group without AGEs treatment, defined as “1”. F Amino acid sequence alignment of β-catenin Y142 residues among β-catenin homologs from different species. Y142 residue in various species is colored in red. G HUVECs were treated with AGEs (100 μg/ml) for 1 h and then phosphorylation of β-catenin Y142 was measured by western blot. n = 5. Data are shown as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001