Fig. 4.

APE1/Ref-1 regulates LPS-mediated NF-κB signaling without affecting IKK activity. a, b Primary cultured astrocytes were transfected with p2xNF-κB-Luc and pRSV-β-gal with expression plasmids for the flag-tagged APE1/Ref-1 (wild-type- and ΔNLS-Ref-1) or APE1/Ref-1 short interfering RNA (siRef-1; 100 pmol). c Primary cultured astrocytes were transfected with the expression plasmids of the p2xTRE-Luc, pRSV-β-gal, WT-, and ΔNLS-Ref-1. After 24 h of transfection, cells were treated with LPS (1 μg/mL) for 6 h. d 293/TLR4/IL-1R cells were transiently transfected with the expression plasmids indicated, along with p2xNF-κB-Luc and pRSV-β-gal. Luciferase assays were performed as described in the Methods, and the activity of each sample was normalized according to the β-galactosidase activity. Each column shows the mean ± SEM of at least three independent experiments. *p ˂ 0.05 vs. mock-transfected cells. e, f 293/TLR4/IL-1R cells were transiently transfected with either wild-type- or ΔNLS-Ref-1 for 24 h, and then cells were treated with LPS for the times indicated. Whole-cell lysates were immunoblotted with the indicated antibodies (e). Cell extracts were subjected to immunoprecipitation with an anti-IKK-γ antibody, and its activity was assessed by an immune complex kinase assay, as described in the Methods (f, upper panel). The specificity of the immunoprecipitation of the IKK complex was confirmed by immunoblotting with an anti-IKK-γ antibody (f, lower panel)