GST fusion proteins CTCF WT and mutant (T374E and S402E) (ZF4-ZF5: amino acids 351 to 410) were generated by PCR, cloned into GST tag bacterial expression vector pGEX-KG, and verified by DNA sequencing and then transformed into BL21 competent cells for protein expression. Protein expression was induced using 0.1 mM isopropyl β-d-1-thiogalactopyranoside and subsequent incubation at 18°C overnight. Protein samples were purified using glutathione-conjugated sepharose and stored at −80°C until use. The homogeneity and concentration of the proteins were estimated by SDS–polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining with bovine serum albumin (BSA) as a standard control.

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