Figure 2. Tests for the interaction between ENY2 and dCTCF in vitro. (A) Cross-linking of ENY2 by incubation with increasing concentration of glutaraldehyde (GA). Proteins were separated in 5–12% gradient polyacrylamide gels and visualized by silver staining (for experimental details, see Materials and Methods). GST was used as a positive control of dimerization. Thioredoxin used as a negative control is shown presented as a monomer molecule. (B) ENY2 interacts with parts of ENY2 in the yeast two-hybrid assay. (C) ENY2 interacts with zinc fingers 7–11 of dCTCF in co-expression assay. Indicated MBP-fused zinc-finger domains were co-expressed with 6 × His-ENY2. The results of 6 × His (stained with Coomassie) and MBP (stained with Coomassie/anti-ENY2 antibodies) pull-down assays are shown. “Input” refers to bacterial lysate. For Sgf11–ENY2, the results of GST and 6 × His pull down assays stained with Coomassie are shown. Arrows indicate positions of MBP-fused zinc fingers of dCTCF or Zw5, GST-fused N-terminus of Sgf11, and 6 × His-ENY2. The zinc-finger domain of Zw5 protein (negative control) displayed no direct interaction with ENY2. The Sgf11 protein (positive control) interacted with ENY2.