Figure 5.

Nuclear BK channel expression is induced by LPS in BV-2 cells. A, BV-2 cells were treated with LPS (100 ng/ml) for 0, 6, 12, or 24 h, fixed, and immunostained with anti-BK antibody (green, BK) and Hoechst 33258 (blue, nucleus), and then were analyzed by confocal fluorescence microscopy. Scale bar, 40 μm. Insets are enlarged views of the selected rectangle areas, and scale bar represents 20 μm. B and C, nuclear, cytosolic, and whole-cell proteins were extracted and immunoblotted. Quantification performed by densitometric analysis of protein bands was normalized to either lamin B (nuclear BK, C left) or β-actin levels (cytosol BK, C middle; whole-cell BK, C right). D, BV-2 cells were untreated (control) or treated with LPS (100 ng/ml) for 12 h in the absence or presence of either Bay11-7082 (20 μm, specific NF-κB inhibitor) or CLI095 (2 μm, specific TLR4 inhibitor). Quantification performed by densitometric analysis of protein bands was normalized to β-actin levels. Data are presented as scatter plots; bars represent mean ± S.D. of five (Nuclear BK, C left; cytosol BK, C middle; and whole BK, D right) or three (whole BK, C right) independent experiments, each performed once. Differences among the groups were analyzed by one-way ANOVA with Newman-Keuls' multiple comparisons post hoc test (C and D). F(3,16) = 13.15 (p = 0.0001) for C left, Nuclear BK; F(3,16) = 1.694 (p = 0.2082) for C middle, Cytosol BK; F(3,8) = 6.723 (p = 0.0141) for C right, whole BK; F(3,16) = 4.894 (p = 0.0134) for D. *, p < 0.05; ***, p < 0.001 compared with the control group. #, p < 0.05 compared with the LPS group.