Figure 3

Effects of ent -Sauchinone on NF-κB DNA binding activity and protein expressions of NF- κB in astrocytes and microglial BV-2 cells. Astrocytes (A) and microglial BV-2 cells (B) were treated with 1 μg/ml of LPS alone, or with LPS plus different concentrations (1, 5, and 10 μM) of ent-Sauchinone at 37°C for 1 hour. Activation of NF-κB was investigated using EMSA as described in Materials and Methods. Nuclear extracts from astrocytes treated with LPS alone (1 μg/ml) or with ent-Sauchinone (1, 5, and 10 μM) and LPS were subjected to DNA binding reaction with ³²P end-labeled oligonucleotide specific to NF-κB. Specific DNA binding of the NF-κB complex is indicated by an arrow. Similar results were obtained from at least three different sets of experiments. Equal amounts of nuclear extract (40 μg) were subjected to 10% SDS-PAGE. Nuclear translocation of p50 and p65, and degradation of IKKα and IκB were detected by western blotting using specific antibodies. β-Actin protein was used here as an internal control (C and D). Values represent the mean ± S.E. for three independent experiments performed in triplicate, and each luciferase activity was calibrated using the amount of protein. Values above of each figure show the quantified relative expression of the proteins or DNA binding activity.