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. 2015 Sep 23;6(5):79–90. doi: 10.1080/21541264.2015.1095269

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© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The moral rights of the named author(s) have been asserted.

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Figure 4. — CTCF interacts with NELF, DSIF, and P-TEFb. (A) Proteins in HeLa nuclear extract were pulled down using nickel agarose beads previously incubated with his-tagged recombinant CTCF (his-CTCF). The bottom panel shows results obtained with more stringent wash conditions (300 mM NaCl vs. 150mM NaCl for the top panel). Western blot analysis was performed using the indicated antibodies. Nuclear extract (NE) incubated with beads without his-CTCF was used as a negative control. Five percent of NE used for the assay was loaded. (B) After incubation with his-CTCF, recombinant FLAG-tagged NELF and DSIF were pulled down using anti-FLAG agarose beads followed by western blot analysis using the indicated antibodies. Incubation of his-CTCF with the FLAG peptide was used as a negative control and 30% of his-CTCF used for the assay was loaded. (C) Recombinant his-CTCF was pulled down using glutathione sepharose beads previously incubated with GST (as a negative control) and GST-tagged Nelf-A and Nelf-E. Western blot was performed using CTCF antibodies and recombinant proteins were detected by SDS-PAGE followed by Coomassie staining. Fifty percent of his-CTCF used for the assay was loaded.