Fig. 4.

NF-κB activation decreases Bmp2-stimulated Runx2 and Wnt-stimulated β-catenin binding to osteocalcin or Bsp promoters. Cells were incubated with Bmp2 or Wnt3a for 4 hours with or without a 45-minute pre-incubation with TNFα. Some of the cells were incubated with NF-κB small molecule inhibitor (BAY-117082). Cells were fixed, DNA isolated, and subjected to ChIP using antibodies for Runx2, β-catenin, RNAP-II or control IgG. (A, B) RT-PCR was performed using primers to Bsp or OC promoter that contained NF-κB response elements. (C, D) Immunoblotting with antibody to beta-catenin, Runx2, actin or laminin of nuclear or cytosolic lysates of cells incubated with Wnt or Bmp for 4 hours, followed by a 3-hour cycloheximide incubation. Before stimulation, some of the cells were incubated with TNFα for 45 minutes. (E) ChIP assay was performed with antibody to RNAP-II or control IgG. RT-PCR was performed using primers to Bsp or OC promoter that contained binding regions of RNAP-II. *Significantly different from No-Bmp2/Wnt3a-stimulated control; +significant inhibition with TNFα; #significantly different between cells incubated with no inhibitor (p < 0.05).