Flag tagged-CTCF (WT and mutant) proteins from transfected HEK293 cells were obtained by immunoprecipitation with anti-Flag antibodies, followed by elution with Flag peptides. The purified proteins were then incubated with biotin-labeled double-stranded DNA oligos [~70 base pairs (bp)] carrying a CTCF-binding site from the AXL gene and then pulled down with reaction buffer [50 mM tris (pH 8.5), 50 mM KCl, 5 mM MgCl2, 0.5% BSA, and 5% glycerol] containing 0.1% NP-40 for 1 hour at 4°C, and then mixed with streptavidin magnetic beads. The beads were washed extensively with the reaction buffer and were analyzed by SDS-PAGE and Western blotting with anti-CTCF antibody. Western blotting followed standard molecular biology procedures. Quantifications of Western blots were performed with ImageJ and reflected the relative amounts as a ratio of phospho-CTCF protein band relative to the lane’s loading control.

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