Figure 4.

Berteroin inhibits nuclear factor (NF)-κB translocation to the nucleus and its DNA binding activity in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. The cells were treated with various concentrations of berteroin for 40 min. LPS was added, and the incubation was continued an additional 20 min. (A) Cytosolic and nuclear fractions were prepared 20 min after the incubation with LPS, and the levels of p65 were analyzed by Western blotting. Photographs of chemiluminescent detection of the blots, which were representative of three independent experiments, are shown (upper panel). Relative density (mean ± SEM, n = 3) was calculated using Image J software (lower panel). Levels of α-tubulin and lamin B were used as cytosolic and nuclear loading controls, respectively; (B) The effect of berteroin on the DNA binding activity of p65 was evaluated by electrophoretic mobility shift assay. The nuclear extracts were incubated with the [γ-³²P]-labeled NF-κB consensus oligonucleotides for 30 min. Each sample was subjected to 5% nondenaturing gel electrophoresis. (Left) An autoradiograph of the dried gel, which was representative of three independent experiments, is shown. (Right) The relative abundance of each band was quantified, and the LPS control levels (1 mg/L LPS + 0 μmol/L berteroin) were set to 100%. Each bar represents the mean ± SEM (n = 3). * Significantly different from the control group (p < 0.05). Means with different letter (a, b, c or d) are significantly different among the berteroin groups (p < 0.05).