FIG. 2.

Cytoplasmic localization of NF-κB induced by E₂. (A) Confocal microscopy images of fluorescence immunocytochemical analysis of p65 (green) in RAW 264.7 cells treated with 1 nM E₂ for 10 min and with LPS for 30 min. Nuclei were counterstained with Sytox orange (red). ctrl, control. (B) Quantification of the effect of E₂ on p65 intracellular localization. The percentage of cells with a cytoplasmic localization of p65 is plotted relative to the total cell number. L, LPS. (C) Western blot analysis of p65, β-actin, and histone H1 in cytoplasmic extracts (Cyt. E.) and nuclear extracts (N. E.) from cells treated as described for panel A. (D) c-Rel subcellular localization. The subcel-lular localization of c-Rel in RAW 264.7 cells treated with 1 nM E₂ for 10 min and with LPS at 50 μg/ml for 30 min was assessed by immunocytochemical (ICC) analysis with anti-c-Rel antibody. (E) Quantification of the effect of E₂ on c-Rel intracellular localization. The percentage of cells with a cytoplasmic localization of c-Rel is plotted relative to the total cell number. (F) Western blot analysis of p50 in nuclear extracts (N. E.) from RAW 264.7 cells treated with a cytokine mixture (CM) for 30 min and with E₂ added before the CM. In panels B and E, values are the means ± SDs of a single experiment performed in triplicate. Single and double asterisks indicate P values of <0.05 in a comparison with the control and in a comparison with LPS, respectively.