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Measurement of O-GlcNAcylated endothelial nitric oxide synthase by using 2′,5′-ADP-Sepharose pull-down assay

Long, Yang, Yan, Jianghong, Luo, Suxin, Liu, Zhenguo, Xia, Yong
Analytical biochemistry 2017 v.537 pp. 8-12
N-acetylglucosamine, animal disease models, animal tissues, antibodies, binding capacity, cultured cells, diabetes, endothelial cells, endothelial nitric oxide synthase, precipitin tests, rats, resins, vascular tissues
Endothelial nitric oxide synthase (eNOS) plays central roles in cardiovascular regulation and disease. eNOS function is critically affected by O-linked N-acetylglucosamine (O-GlcNAc) modification. The present method for measuring O-GlcNAcylated eNOS relies on immunoprecipitation. Such method exhibits low detection efficiency and is also costly. We here report a simplified assay by employing the high binding affinity of eNOS with the 2′,5′-ADP-Sepharose resins. Together with the O-GlcNAc antibody, this assay readily allows the detection of O-GlcNAcylated eNOS in both cultured endothelial cells and rat vascular tissues. By using this assay, we demonstrate that eNOS O-GlcNAcylation is markedly elevated in the vessels of diabetic rats. Thus, a 2′,5′-ADP-Sepharose-based pull-down assay is developed to measure O-GlcNAcylated eNOS. This assay is simple and efficient in detecting O-GlcNAcylated eNOS in cultured cells and animal tissues under both normal and disease conditions.