FIGURE 5.

Lys-717 and Lys-719 in ANK7 of IκBζ are crucial for association with the Lcn2 promoter DNA. A and D, GST-fused IκBζ-(414–728) or a mutant IκBζ carrying the indicated substitution were incubated with His-p50 in the presence of WT Lcn2 (−317/−117) or a mutated κB site (κBm)-carrying Lcn2 (−317/−117). After the protein-DNA complex was pulled down with glutathione-Sepharose-4B beads, the co-precipitated DNA was amplified by PCR, and the product was analyzed by agarose gel electrophoresis. The precipitated proteins were subjected to SDS-PAGE, followed by staining with CBB or immunoblot with anti-His antibody. MW, molecular weight. B, formaldehyde-fixed chromatin was prepared from RAW264.7 cells stably expressing FLAG-IκBζ (WT), FLAG-IκBζ (K717E/K719E), or FLAG-IκBζ (D451T) (top panel) and subjected to ChIP assay using anti-FLAG (M2) mouse monoclonal antibody or anti-RNA polymerase II antibody (bottom panel). Precipitated DNA was analyzed by PCR using primers corresponding to the Lcn2 locus. The results are representative of experiments from at least three independent experiments. IP, immunoprecipitation. C, the role of Gly-718, Ser-720, and Ile-721 in Lcn2 activation. p50-/IκBζ-deficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Lcn2 (−1031/+54), the internal control plasmid pRL-TK, and pcDNA3 for expression of FLAG-IκBζ with the indicated amino acid substitution and HA-p50. Luciferase activities were determined as described under “Experimental Procedures.” Each graph represents the mean ± S.D. obtained from three independent transfections. Cell lysates were analyzed by immunoblot with anti-FLAG, anti-HA, or anti-β-tubulin antibody. Positions for marker proteins are indicated in kilodaltons.