Characterization of biotinylated CCCTC binding factor (CTCF). (A) Identification of CTCF-binding partners. CTCF (light grey boxes) was tagged with biotin at its C-terminus (biotinylated lysine indicated by a star) and pulled down, and interacting proteins were identified by mass spectrometry. Zinc fingers are indicated by dark grey box; proline-rich region with AT hook. (B) Targeting of mouse Ctcf. The Ctcf gene is shown on the top line. Exons (black boxes are coding, open boxes are non-coding) and probes (1, 2) are indicated. X = Xba I, P = Pst I. Targeting construct is shown in the middle. Bio = biotin-tag, black triangles = loxP sites, Neor = PMC1-neomycin cassette. After targeting, Ctcfbio-neowas obtained. Cre-mediated excision yielded the Ctcfbioknock-in allele (bottom line). (C) Southern blot analysis. Digested embryonic stem (ES) cell DNA was analyzed by Southern blotting using probes 1 or 2. Alleles are indicated. (D) PCR analysis of mouse tail DNA. (E) Expression of biotin tagged CTCF (CTCF-bio). Nuclear thymic extracts were analyzed by western blotting. Coomassie staining shows total protein. (F) CTCF-bio pull-down assay with streptavidin beads. Nuclear ES cell extracts were incubated with streptavidin-coupled magnetic beads. Input (i) = 5% of nuclear extract, (u) = 6% of unbound fraction, b = material bound to beads. (G) Size fractionation of CTCF and CTCF-bio. Size fractionated nuclear extracts were analyzed by western blotting. Molecular mass markers are indicated. V0 = void volume, input = nuclear extract (5%). (H) Chromatin immunoprecipitations. CTCF-bio precipitated with streptavidin-coupled magnetic beads from formaldehyde fixed nuclei bound known CTCF sites (β-globin 3" HS1 and c-Myc insulator).