CoIP assays demonstrating physical interaction of Pax8 with Sp1 and CTCF. A) Nuclear extracts from control or hCTCF-transfected PCCl3 cells were obtained and immunoprecipitated (IP) with anti Pax8, anti-Sp1 or anti-CTCF antibodies. Immunoblotting was performed against Sp1 (top, left panel), CTCF (top, right panel) or Pax8 (bottom, left and right panels). Lanes 1 are the input and lanes 2 are the nonspecific IPs using IgG. The Figure shows a representative Western-Blot. B) Reporter assays were performed using the pNIS-2.8 promoter and expression vectors as indicated in the figure. Promoter activity is expressed as fold induction, relative to the activity observed in the presence of empty expression vector. The amount of total DNA used for each transfection was adjusted with the matched empty vector control to 1 μg. Luciferase activity was normalized to renilla activity derived from the cotransfected pRL-CMV vector to adjust for transfection efficiency. Results are mean ± SD of three independent experiments. * p-value < 0.005; **, p-value < 0.01.