Figure 4

Inhibition of the nuclear accumulation of the NF-κB p65 subunit and degradation of IκB-α by ATL in LPS-stimulated BV-2 microglial cells. (A) BV-2 cells were stimulated with 100 ng/ml LPS for the indicated times. Subcellular localization of p65 subunit was evaluated using an anti-p65 antibody and a FITC-labelled anti-rabbit IgG antibody. DNA was stained using DAPI to visualize nuclei, and cells were visualized using laser confocal scanning microscopy. Note that nuclear translocation of the p65 subunit is not complete, but that part of the cytoplasmic p65 is translocated to the nucleus so that the distinction between the nucleus and the cytoplasm blurs. This is obvious 60 min after activation. (B) BV-2 cells were stimulated with 100 ng/ml LPS in the absence or presence of 100 nM ATL that had been added 30 min before activation. Subcellular location of the p65 subunit was tested using immunofluorescence assay 60 min after activation. (C) BV-2 cells were stimulated as in B. Cytoplasmic and nuclear extracts were separated by SDS-PAGE and immunoblotted with anti-p65 antibody. The same extracts were re-electrophoresed and immunoblotted for β-actin or lamin B to monitor loading. A representative result from three independent experiments is shown. (D) Quantification of cytoplasmic and nuclear p65 bands from the experiments in C was normalized by β-actin or lamin B. (E) BV-2 cells were pretreated with vehicle or 100 nM ATL for 30 min and stimulated with LPS (100 ng/ml). Levels of IκB-α in cellular lysates were analyzed using western blotting at indicated times. Quantification of IκB-α protein levels was performed by densitometric analysis. Data are presented as mean ± SEM for three independent experiments.*P < 0.05 compared with LPS in the absence of ATL;**P <0.01 compared with LPS in the absence of ATL; ^## P < 0.01 compared with vehicle.