The IKK complex is involved in PMA-induced p65 phosphorylation and nuclear localization and c-Fos expression.

<p>In most experiments, WT, IKKα^−/−, IKKβ^−/−, IKKγ^−/−, and p65^−/− MEFs were treated with TNF-α (20 ng/mL) or PMA (10 nM) for the indicated times. (A, D, F) Total cell lysates were prepared and subjected to western blotting analysis; the antibodies used are indicated. The western blots are representative of 3 independent experiments. (B) Total RNA was extracted, reverse transcribed into cDNAs, and subjected to qPCR analysis. Values were normalized relative to β-actin gene expression and expressed relative to the control sample. * <i>P</i><0.05 indicates a significant reduction in PMA-induced gene expression in IKK-null MEFs. (C) Distinct response elements and the predicted NF-κB-binding site in the mouse <i>c-fos</i> promoter are shown. Regulatory elements identified in the mouse <i>c-fos</i> promoter are labeled and include the cis-inducible elements SRE, Fos activator protein 1 site (FAP), a glucocorticoid response element (GRE), and CRE. The major CRE is nearest of the sites to the start site within the large box, whereas the minor CREs are further upstream and are denoted using small boxes. Constitutive activity of the <i>c-fos</i> promoter with or without mutation of the κB site was determined in WT and IKK-null MEFs. Data in (B) and (C) are the means ± SEM from 3 independent experiments. * <i>P</i><0.05 indicates a significant reduction in basal and PMA-induced promoter activity in IKK-null MEFs. (D) The p65^−/− MEFs were reconstituted with p65 protein, and HEK293 cells were transiently transfected with control or p65 siRNA (100 nM) for 48 h. Cells were treated with PMA for the indicated times. (E) Immunofluorescent staining of p65 (green) and nuclei (blue) shown by images captured under a fluorescence microscope; similar results were obtained in 2 additional experiments.