Figure 3.

Modulation of O-GlcNAc and the transcriptional activity of c-Rel induced by stimulation with LPS with or without GlcN. (A, B) BV2 cells were untreated or pretreated with GlcN (5 mM) for 2 h and stimulated with LPS (0.1 μg·mL⁻¹) in the presence or absence of 5 mM GlcN for 24 h. Total cell lysates (Input) were prepared and subjected to precipitation (IP) with WGA-conjugated agarose beads. Precipitated proteins were immunoblotted by c-Rel, p50, O-GlcNAc (CTD110.6) or GAPDH antibodies (A). Cell lysates were immunoprecipitated with anti-c-Rel antibody. Immunoprecipitates were subjected to galactosyltransferase labelling using [³H]-UDP-galactose as a substrate (B). (C) RAW264.7 cells were transfected with pFR-luc promoter containing five GAL4 binding sites in its promoter with or without c-Rel protein fused to GAL4 DBD. Cells were then treated with 5 mM GlcN with or without LPS (0.1 μg·mL⁻¹) for 24 h and luciferase activity, in relative light units, was measured. Data are representative of three independent experiments. *denotes P < 0.01 compared with LPS-treated samples.