Figure 2. TNF alpha induces binding of NFkappaB and C/EBP to the Met promoter.

(A) The nucleotide sequence of the Met promoter region corresponding to −745 to -697 bp from the transcription start site is shown (top). It contains a putative NFkappaB site (blue oval/sequence in blue font) and tandem C/EBP sites (upstream C/EBP site [uC/EBP] – red oval/sequence in red font; palindromic C/EBP site [pC/EBP] – green oval/sequence in green font). The arrows over the pC/EBP site indicate the palindromic repeats. Oligonucleotides used in various experiments are shown (below). Yellow font indicates sequence that was mutated in mutant oligomers. (B) The S1 oligonucleotide (a 50-mer) corresponding to the Met promoter composite site was used as a probe in EMSAs. Nuclear protein extracts (NPE) from untreated control (lanes 2-8) or TNF alpha treated Hepa1-6 cells (lanes 9-15) were added as indicated. For supershift assays, various antibodies (Ab) against the NFkappaB family (p65 [65], p50 [50], and c-Rel [Rel]) as well as anti-C/EBP alpha (al), C/EBP beta (be), and C/EBP delta (de) antibodies were utilized as indicated in the figure. Oligonucleotides corresponding to the published consensus C/EBP (C) and NFkappaB (D) binding sites (labeled CC and CN in Figures 2C and D, respectively) were used as probes in gel shift assays with nuclear protein extracts (NPE) from Hepa1-6 cells that were treated with or without TNF alpha. Various unlabeled oligos derived from the Met promoter composite element (i.e. S2, M2, S3, M3 – see [A] for sequence) were added as competitors as indicated in the figures. A variety of antibodies (Ab) as described in [B] were used in supershift studies as indicated. NS – non-specific negative control oligomer; mN – mutant concensus NFkappaB site oligomer; Ig – IgG negative control antibody; p65 – binding complex containing p65; p50 – binding complex containing p50.