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. 2016 Aug 3;291(39):20739–20752. doi: 10.1074/jbc.M116.719302

FIGURE 3.

FIGURE 3.

Asp-451 of IκBζ and Asp-127 of Bcl-3 participate in transcriptional activation. A and D, the role of IκBζ Asp-451 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, pcDNA3 for expression of FLAG-IκBζ (WT) or FLAG-IκBζ (D451T), and HA-p50 (A) or HA-p52 (D). 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. MW, molecular weight. B, IκBζ-mediated activation of the endogenous Lcn2 gene. IκBζ-deficient BMMs were retrovirally transduced for expression of WT IκBζ or a mutant protein with the D451T or K717E/K719E substitution. Proteins in the cell lysate were analyzed by immunoblot with the anti-IκBζ or anti-β-tubulin antibody (left panel). The transduced BMMs were stimulated for the indicated time with LPS, and the relative amounts of mRNA transcribed from the endogenous Lcn2 gene were estimated by quantitative real-time RT-PCR as described under “Experimental Procedures” (right panel). Each graph represents the mean ± S.D. in triplicate determinations. C, the role of Bcl-3 Asp-127 in Ccl2 activation. RAW264.7 cells were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Ccl2 (−2777/+76), the internal control plasmid pRL-TK, and pcDNA3 for expression of FLAG-Bcl-3 (WT) or FLAG-Bcl-3 (D127T) and HA-p52. 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-actin antibody. E, association of p50 with the Lcn2 gene promoter. His-tagged p50 (WT), p52 (WT), p65 (WT), or p50 (Y57A/E60D) was incubated with the Lcn2 gene promoter (−317/−117). After the protein-DNA complex was pulled down with COSMOGEL® His-Accept, the co-precipitated DNA was amplified by PCR, and the product was analyzed by agarose gel electrophoresis. The precipitated proteins were subjected to immunoblot analysis with anti-His antibody. Positions for marker proteins are indicated in kilodaltons.