Interaction of CCCTC binding factor (CTCF) and CTCFL with upstream binding factor (UBF). (A) Biotin tagged CTCF (CTCF-bio) interacted with UBF and RNA polymerase I in embryonic stem (ES) cells. Nuclear extracts from ES cells expressing CTCF-bio and control ES cells (-) were incubated with streptavidin beads, and CTCF-bio was purified with interacting proteins. Extracts were treated with (+) or without (-) benzonase for 2 hours at 4°C. Western blots were incubated with the indicated antibodies (CTCF-bio detected with streptavidin-coupled horseradish peroxidase). UBF was detected as a doublet consisting of UBF1 and UBF2; RPA194 = the large subunit of RNA polymerase I. B = bound fraction; i = input (5%). (B) Both CTCF and CTCFL interacted with UBF. Cells were transfected with different cDNAs (indicated below the lanes). CTCFL was not tagged. Immunoprecipitations (IPs) were carried out using anti-Flag antibodies. Western blots were incubated with antibodies against the indicated proteins. (C) Green fluorescent protein (GFP) tagged CTCF deletion mutants. The regions of CTCF used for making the different fusion proteins are indicated by lines. (D) UBF interacts with the zinc-finger domain of CTCF. GFP tagged CTCF deletion mutants (in (C)) were co-expressed with Flag tagged UBF in HEK293T cells. All fusion proteins are expressed at similar levels (input). After a Flag pull-down (IP), co-precipitating proteins were detected with an antibody against GFP. Lane numbers correspond to mutant numbers in (C).