Fig. 4

Attenuation of LPS-induced NF-κB activation by HHS in RAW 264.7 macrophages. a The cells were treated with the indicated concentrations of HHS for 1 h before LPS treatment (100 ng/ml) for 30 min. Nuclear and cytosolic proteins were resolved on 10 % SDS-polyacrylamide gels, followed by Western blotting using anti-NF-κB p65 and anti-IκBα antibodies. Lamin B and actin were used as internal controls for the nuclear and cytosolic fractions, respectively. b Cells were pretreated with 300 μg/ml of HHS for 1 h prior to stimulation with LPS (100 ng/ml) for 30 min. Localization of NF-κB p65 was visualized with a fluorescence microscope after immunofluorescence staining with anti-NF-κB p65 antibody and an FITC-labeled anti-rabbit IgG antibody (green). Nuclei of the corresponding cells were visualized with DAPI (blue). c Following treatment with 300 μg/ml of HHS for 1 h, the cells were treated with 100 ng/ml of LPS for 30 min. Nuclear extracts were prepared, and the DNA binding activity of NF-κB was analyzed by an EMSA