FIGURE 3.

CTCF interacts with the condensin complex both in vivo and in vitro. A, two-step purification schematic for identifying CTCF-associated proteins. B, immunopurified human FLAG-CTCF complexes were fractionated on SDS-PAGE. C, mass spectrometry analysis identifying peptide sequences of SMC2 and SMC4 in the CTCF complex. D, HeLa-S3 nuclear extract with human FLAG-CTCF or FLAG alone were tested for coimmunoprecipitation. The precipitates were analyzed by immunoblot analysis, with antibodies shown on the right. E, GST pull-down assays confirmed that CTCF interacts with SMC2 and SMC4. GST-CTCF and GST fusion proteins were expressed and purified in BL21. F, MCF-7 NEs were precipitated with the antibodies shown at the top, and precipitates were immunoblotted with the antibodies shown on the right. G, CTCF interacts with both condensin I and condensin II. MCF-7 NEs were precipitated with the antibodies shown at the top, and precipitates were immunoblotted with the antibodies shown on the right. CAP-D2 and CAP-D3 are the specific subunits of condensin I and condensin II, respectively. H, DNase and RNase treatment have no effect on the interaction between CTCF and condensin.