Measurement Of O-GlcNAcylated Endothelial Nitric Oxide Synthase By Using 2?,5?-ADP-Sepharose Pull-down Assay

Objective:To investigate a simplified assay to measure O-GlcNAcylated endothelial nitric oxide synthase by employing the high binding affinity of eNOS with the 2?,5?-ADP-Sepharose resins and its application in aortas of type 2 diabetic rats.Methods: Detect the efficiency of 2?,5?-ADP-Sepharose in pulling down eNOS,Measurements of O-GlcNAcylated eNOS by 2?,5?-ADP-Sepharose pull-down assay in cultured endothelial cells,Using 2?,5?-ADP-Sepharose pull-down assay to detect O-Glc NAcylated e NOS in rat aortas,Establish the type 2 diabetic rats model,Alterations of e NOS O-GlcNAcylation in diabetes.Result:The eNOS pull-down efficacy of a single 2?,5?-ADP-Sepharose incubation was estimated greater than 80%,2?,5?-ADP-Sepharose pull-down in combination with anti-O-Glc NAc immunoblotting could effectively detect O-GlcNAcylated e NOS incultured cells and vascular tissues,significantly elevated O-GlcNAcylated e NOS(P>0.05)was detected in the aortas of diabetic rats in comparison to that of normal ones.Conclusion:compared to the immunoprecipitation,This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions.