Figure 2.

Inhibition of the binding of c-Rel to the NF-κB site of an iNOS promoter induced by GlcN. BV2 cells were untreated or pretreated with 5 mM GlcN for 2 h and stimulated with LPS (0.1 μg·mL⁻¹) with or without 5 mM GlcN for 24 h. (A) Nuclear extracts were prepared and loaded (Input: 20 μg each). Binding of c-Rel to the iNOS promoter-derived, biotinylated NF-κB probe was measured by streptavidin-agarose pull-down assay (Pulldown-NF-κB) (as described in Methods) followed by Western blotting for c-Rel. (B) Chromatin DNA was immunoprecipitated with anti-c-Rel antibody and eluted DNA was quantified by real-time PCR or standard reverse transcriptase PCR (RT-PCR) for the iNOS promoter. Input represents each PCR product obtained from 2% of the pre-immunoprecipitated DNA. The real-time PCR data are expressed as mean ± SEM (error bars). The RT-PCR and Western blots shown are representative of three or more independent determinations. *Denotes significantly different from LPS-treated (P < 0.01). qPCR, quantitative PCR.