1. CTCF Tethers an Insulator to Subnuclear Sites, Suggesting Shared Insulator Mechanisms across Species 
Timur M. Yusufzai, Hideaki Tagami, Yoshihiro Nakatani, Gary Felsenfeld 
Molecular Cell 
Volume 13, Issue 2, Pages (January 2004)
DOI: /S (04)
Copyright © 2004 Cell Press Terms and Conditions

2. Figure 1 Purification of CTCF Complexes
(A) FLAG-HA-tagged CTCF was purified from a stably transduced HeLa cell line, visualized by Coomassie staining, and copurifying factors were identified by mass spectrometry.
(B) FLAG-purified CTCF material was fractionated on a 10%–30% glycerol gradient and fractions were collected and analyzed by Western blotting. Note: the lower band in the nucleophosmin Western blotting represents a degradation product. Fractions 5–7 and 12–14 were pooled separately and purified on anti-HA resin. Eluted proteins were then resolved and silver stained.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions

3. Figure 2 Self Interaction of CTCF
(Left) Purified CTCF was resolved by PAGE in the presence or absence of DTT in the loading buffer, followed by silver staining. (Right) A yeast two-hybrid analysis with CTCF as the bait (CTCF-BD) and target (CTCF-AD) was used to determine if CTCF can interact with itself in vivo. Two colonies of each yeast strain carrying both CTCF-BD and CTCF-AD or the fusions in combination with the empty Gal4 binding (BD) and activation domains (AD) as controls were plated on selection media as indicated and allowed to grow for 3 days.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions

4. Figure 3 Specificity of Nucleophosmin Interactions
(A) Purification of nucleophosmin. (Left) FLAG-HA-tagged nucleophosmin was purified by anti-FLAG/HA resins and analyzed by silver staining. (Right) Western blotting of the purified material was done with anti-CTCF, HA, or nucleophosmin antibodies. Both tagged and endogenous nucleophosmin as well as CTCF were detected following the purification.
(B) Controls for specificity of the nucleophosmin interaction. (Left) Control FLAG purifications from wt HeLa cells and cells carrying FLAG-HA-tagged HP1α or PcG (Mel18) followed by anti-HA and antinucleophosmin Westerns. (Right) To test the stability of the CTCF-nucleophosmin interaction, CTCF was also FLAG/HA purified with a 400 mM KCl wash step. CTCF and nucleophosmin were then detected by Western blotting.
(C) In vivo association of nucleophosmin and HS4. (Left) Chromatin immunoprecipitation (ChIP) of nucleophosmin and CTCF with formaldehyde crosslinked chromatin from 6C2 cells and PCR of the chicken β-globin domain. (Right) Silver staining of a control IP of CTCF from uncrosslinked HeLa cells with binding and wash conditions used for the ChIP. All uncrosslinked factors appear to be removed.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions

5. Figure 4 Localization of Insulator-Containing Transgenes
(A) Nuclei from K562 cell lines containing multiple copies of a transgene with wild-type (wt) or mutant (CTT) CTCF sites were fixed and hybridized with a biotin-labeled plasmid probe to determine transgene localization (arrowheads). Pictures of nuclei were taken with a Nomarski filter and a fluorescent filter and overlaid. Nuclei from each cell line were counted and results are summarized in the table.
(B) ChIP on a wild-type (wt) and a CTT mutant line (used in [A]) for CTCF and nucleophosmin.
(C) Model of CTCF tethering of insulator DNA to the nucleolar periphery. CTCF binds to the outer part of the nucleolus and anchors the insulator DNA separating the enhancer (E) from the promoters (P). Only the green promoters can interact with the enhancers.
Molecular Cell  , DOI: ( /S (04) )
Copyright © 2004 Cell Press Terms and Conditions