Figure 4

TBX3 inhibits NFKBIB promoter activity. A) Luciferase reporter assay. COS-7 cells were transfected with either pcDNA3.1-Myc (control) or pcDNA3.1-Myc-TBX3 expression vectors together with the pGL3-NFκBIB luciferase reporter construct and a β-galactosidase control plasmid (pcDNA3.1/His/LacZ). Cell lysates were harvested 48 hours after transfection and used to measure luciferase activity. β-galactosidase enzyme activity was also measured and used to normalize luciferase activity. Error bars indicate SD; n = 3. **, P < 0.001. B) Immunohistochemical staining of Nfκbib in doxycycline induced and un-induced double transgenic mice at 10 weeks of age. The third mammary glands were fixed and embedded in paraffin. Five micrometer thick sections were blocked with hydrogen peroxide and incubated with rabbit anti-Nfκbib antibody overnight. Biotinylated goat anti-rabbit IgG was used as a secondary antibody. Standard ABC kit and DAB kit were used for visualization. Nfκbib was down-regulated in the doxycycline induced double transgenic mouse as compared to its un-induced double transgenic littermate control. Images were captured at 40× magnification.