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. 2010 Mar 10;285(19):14088–14100. doi: 10.1074/jbc.M109.017129

FIGURE 6.

FIGURE 6.

Binding of nuclear complexes to the κB and C/EBP elements of the LCN2 promoter. Nuclear extracts from A549 cells that were either not stimulated (unstim.) or stimulated with IL-17 (200 ng/ml) and TNF-α (20 ng/ml) for 1½ h (IL-17 + TNF-α) were used for EMSA. A, a 32P-labeled probe containing the κB element of the LCN2 promoter was incubated with either no competitor (no comp.) or a 250-fold excess of an unlabeled oligo, which was identical in sequence to the probe (NGAL κB), only differed by a mutated κB element (NGAL κB*), contained sequences of the C/EBP-1 (C1) or C/EBP-2 (C2) elements of the LCN2 promoter, or carried a consensus κB sequence (cons. κB). Antibodies against p50, p65, C/EBP-β, C/EBP-δ, and C/EBP-ϵ were tested for their ability to cause a supershift of the nuclear complex associated with the probe. Supershifts with the p50 and p65 antibodies are shown with an arrow. The specific band formed on the NGAL-κB probe with the nuclear extract from cytokine-stimulated cells is indicated with an asterisk. B, a probe containing the C/EBP-1 element of the LCN2 promoter (C1) was incubated without antibody (no Ab) or with antibodies against C/EBP-β, C/EBP-δ, C/EBP-ϵ, p50, or p65. Supershift with anti-C/EBP-β is indicated with an arrow. C, an experiment similar to that in B was performed with a probe containing the C/EBP-2 element of the LCN2 promoter (C2). A supershift band was also in this case seen with anti-C/EBP-β (arrow). D, a control for supershift using the CRP oligo as probe. Supershift was observed for anti-C/EBP-β and anti-C/EBP-δ. E, the C1 probe was incubated with either no competitor (no comp.) or a 250-fold excess of an unlabeled oligo, which was identical in sequence to the probe (C1), had a 2-base pair mutation in the C/EBP-binding element (C1*), or contained the wild-type (NGAL κB) or mutated (NGAL κB*) NGAL-κB sequence. F, identical to the experiment in E except for C1 being exchanged by C2. G and H, identical to the experiments in E and F except for the NGAL-κB oligoes being substituted by the C/EBP-containing oligo CPR.