FIGURE 5.
IKKαΔNBD restores NF-κB-dependent transcriptional activity. A, WT, IKKα−/−, IKKαWT, and IKKαΔNBD MEFs were treated with either IL-1α (10 ng/ml) (left) or TNF (10 ng/ml) (right) for the times indicated, and then lysates were immunoblotted using anti-IκBα or anti-tubulin (Tub.) as indicated (right). The same panel of MEFs was treated with either IL-1a (B) or TNF (C) for the times indicated, and then nuclear extracts were prepared for EMSA. Assays were performed using either a consensus NF-κB binding site probe (top) or an Oct1 probe as a loading control (bottom). D, WT, IKKα−/−, IKKαWT, and IKKαΔNBD MEFs were transiently transfected with the NF-κB-dependent firefly luciferase reporter construct pBIIx-luc together with a control Renilla luciferase construct. Twenty-four hours later, cells were either left untreated (Control) or treated with IL-1α or TNF for 5 h, and then NF-κB activity was determined by dual luciferase assay. E, the MEF panel was either untreated (−) or incubated with TNF for 30 min (+), and then whole cell lysates were immunoblotted using anti-phospho-p65 (P-p65), anti-p65, anti-IKKα, and anti-tubulin (Tub.), as indicated (right). F, WT, IKKα−/−, IKKαWT, and IKKαΔNBD MEFs were either untreated or stimulated for 30 min with TNF, and then nuclear (N) and cytoplasmic (C) extracts were prepared and immunoblotted using the antibodies indicated (right). The integrity of the cytoplasmic and nuclear extracts was confirmed using anti-tubulin and anti-histone H3, respectively. G, MEFs were either untreated (−) or stimulated for 30 min with TNF (+), and then nuclear extracts were prepared and immunoblotted using anti-phospho-IKKα/β (P-IKKα/β), anti-IKKα, anti-IKKβ, and anti-histone H3 as indicated (right).