Fig 2.

CTCF binding to the HVS orf73/orf74 promoter region influences promoter activity. (A) Oligonucleotide pulldown with biotinylated DNA from HVS C488 region 107134 to 107724. HEK293T nuclear extract was incubated with biotinylated DNA fragments and bound proteins were identified by Western blotting with an antibody specific for CTCF. (B) Fluorescent electrophoretic mobility shift assay (EMSA) with Cy5-labeled double-stranded DNA fragments encompassing fragment 1 or 2 (same fragments as in panel A), respectively. DNA fragments were incubated in the presence of recombinant CTCF protein (lanes 1 and 2) or in the presence of recombinant CTCF protein and a CTCF antibody (lanes 4 and 5) prior to gel electrophoresis. DNA fragments, fragment shifts, or supershifts were visualized by measuring the fluorescence signal. (C) Luciferase reporter assays. HEK293 cells or OMK cells were transfected with equal DNA amounts of empty vector pGL3 basic or one of the plasmids depicted in the upper scheme. A cotransfected renilla luciferase construct served as the internal standard for transfections. Luciferase assays were performed 1 day after transfection. Bars represent arithmetic means and standard deviations of luciferase activity normalized to renilla activity from three independent transfection experiments.