Figure 2

A. The OPN promoter lacking the NF-κB site is relieved for BRMS1 suppression. The grey box represents the predicted NF-κB site. The dotted line indicates the region deleted in the construct. OPN-ΔI1 does not have the NF-κB-binding site and is not suppressed by BRMS1. OPN/NotI, in which the NF-κB site is abolished by inserting a NotI site in its place, is refractory to suppression by BRMS1. Data is expressed as Relative luciferase activity of control, where control (pGL3-OPN) is 100%. The data shown represents more than three independent experiments in triplicate. * indicates significant suppression, p value < 0.05 and ** indicates p < 0.01 compared to respective controls. B. Recombinant p65 and p50 bind to and retard the mobility of the predicted NF-κB site from the promoter of OPN. Lane 1: Probe bearing the NF-κB site from OPN promoter; Lane 2: Probe + recombinant p50; Lane 3: Probe + nuclear extract of MDA-MB-435; Lane 4: Probe + unlabeled 'cold' consensus probe + Nuclear extract of MDA-MB-435; Lane 5: Probe + recombinant p65; Lane 6: Probe + recombinant p65 + anti-p65 antibody; Lane 7: Mutant probe; Lane 8: Mutant Probe + recombinant p65; Lane 9: Mutant Probe + recombinant p50. The box gives the sequence comparison of the OPN/NF-κB with the consensus NF-κB-binding site. The underscored bases in OPN/NF-κB represent variation from the consensus. Mutant OPN/NF-κB (Mut OPN/NF-κB) is also shown with the mutations represented in bold lower case. C. NF-κB subunits bind to the OPN promoter in vivo . MDA-MB-435 cells were fixed with formaldehyde, lysed, and then sonicated. In vivo cross-linked chromatin was then precipitated independently using p65 (Anti-p65), p50 (Anti-p50), normal rabbit IgG (Rabbit), no antibody, or Positive and Negative kit control antibodies (Active Motif). The recovered immunoprecipitated DNA was then used for PCR with primers specific for the OPN/NF-κB-containing promoter segment. A 151 bp product corresponding to a region 1575 bp upstream of the OPN/NF-kB site (that lacks a predicted NF-κB site [12]) was absent in a control PCR following ChIP Primers: 5'-TTCCCCCTACCAAATGTTCA-3' and 5'-TGCTGCAAAAGTAATTGTGGTT-3'.