Fig. 7. EUE induces nuclear translocation of Nrf2 in BV-2 microglial cells. (A) Time-dependent EUEmediated induction of Nrf2 nuclear translocation. Cells were treated with 100 μg/ml EUE for 0.5, 1, 2, 4, 6, 12, and 24 h. Nuclear fractions were then prepared and assayed for the presence of Nrf2. (B) Concentrationdependent induction of nuclear translocation of Nrf2 by EUE. Cells were treated with different concentrations of EUE for 6 h. Nuclear fractions were then prepared and analyzed for the presence of Nrf2 and lamin B1 by Western blotting. Densitometric results are presented as means ± S.E.M. (n=3). (C) Immunofluorescence analysis of Nrf2 nuclear translocation. Nrf2 was detected with anti-Nrf2 antibodies and Alexa Fluor? 488-conjugated secondary antibodies. Nuclei were counterstained with Hoechst 33258, and representative pictures were captured with a fluorescence microscope (100× magnification). The images shown are representative of three experiments. Scale bar: 200 μm. (D) ARE reporter assay with EUE. Cells were transiently transfected with an ARE reporter construct and then treated with the indicated concentrations of EUE for 6 h. Equal protein amounts of cell extracts were then assayed for dual-luciferase activity. Data are presented as means ± S.E.M. (n=3). (E) EMSA analysis of DNA binding by Nrf2. Cells were pretreated with the indicated concentrations of EUE for 6 h and nuclear extracts were then prepared. The binding of Nrf2 to DNA was investigated by EMSAs. *p<0.05; **p<0.01; and ***p<0.001 compared with the control group.
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