Fig. 5.

COS abolishes LPS-induced NF-κB/p65 O-GlcNAcylation in endothelial cells. BAEC were pretreated with COS (up to 100 μg/ml) for 1 h and then exposed to LPS (100 ng/ml) for 1 h. Western blotting was performed with O-GlcNAc-modified proteins enriched by WGA either from (A) whole cell lysates (the protein loading or input was assessed by Western blotting on whole cell lysates) or (B) nuclear fraction isolated with NE-PER® Nuclear and Cytoplasmic Extraction Reagents (the protein loading or input was assessed by Ponceau S staining of the blot). (C) DNA binding activity was measured by following the instruction of the NF-kB p50/p65 Transcription Factor Assay Kit provided by the manufacture (Abcam). It was achieved by quantification of the binding of NF-κB (from WGA-enriched nuclear extracts) to the DNA containing NF-κB response elements (immobilized on a 96-well plate from the kit). Data are expressed as means ± SEM (n=3). ^##P<0.01 compared to the vehicle-treated control group; **P<0.01 compared to the LPS-only group.