FIGURE 8.

Interaction of IκBζ with the Lcn2 promoter, and its activation requires the 5′-YCCC-3′ sequence downstream of the κB site. A, the 5′ upstream promoter region of the mouse Lcn2 gene. The κB site and its downstream IκBζ-response element in the region are underlined. The nucleotide sequences of the corresponding region from various species are aligned. Mm, Mus musculus; Hs, Homo sapiens; Pa, Papio anubis; Rn, Rattus norvegicus. B–G, base preference of the extra-κB site in Lcn2 activation (top panels) and in formation of the three-species complex containing IκBζ, p50, and promoter DNA (bottom panels). For estimation of Lcn2 activation, p50-/IκBζ-deficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of wild-type Lcn2 (−500/+50); a mutant Lcn2 with the indicated base replacement, or the IκBζ-independent gene Sele (−445/+105); the internal control plasmid pRL-TK; and pcDNA3 for expression of FLAG-IκBζ and HA-p50. Luciferase activities were determined as described under “Experimental Procedures.” Each graph represents the mean ± S.D. obtained from three independent transfections. For estimation of protein-DNA complex formation, GST-IκBζ was incubated with His-p50 in the presence of the DNA fragment of wild-type Lcn2 (−500/+50), a mutant Lcn2 with the indicated base replacement, or Sele (−445/+105). After the 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.