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Interactions between insulator factors.

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posted on 28.08.2014, 03:22 by Jutta Vogelmann, Antoine Le Gall, Stephanie Dejardin, Frederic Allemand, Adrien Gamot, Gilles Labesse, Olivier Cuvier, Nicolas Nègre, Martin Cohen-Gonsaud, Emmanuel Margeat, Marcelo Nöllmann

(A) Co-immunoprecipitation pulldown assay (co-IP) with heterologously purified BEAF32 and Chromator. Goat-IgG or purified guinea-pig polyclonal antibodies against Chromator were covalently coupled to agarose beads. BEAF32 and Chromator were incubated and analyzed by SDS-PAGE followed by Western-Blot-analysis. Lane 1 (input) shows the presence of both BEAF32 and Chromator in the mix. Both proteins are retained by an anti-Chromator column, but not by an anti-goat-IgG column. (B) Co-IP of purified BEAF32 and Chromator-C. Chromator antibody recognizes Chromator and Chromator-C equally well (Materials and Methods). A mix of BEAF32/Chromator was incubated and analyzed by PAGE/Western blotting as before. Both BEAF32 and Chromator-C remain bound to an anti-Chromator column, consistent with the interaction between BEAF32 and Chromator being mediated by its C-terminal domain. (C) Co-IP of purified BEAF32 and CP190. A mix of BEAF32/CP190 was incubated and analyzed by PAGE/Western blotting. Both BEAF32 and CP190 remain bound to a rabbit anti-CP190 column, suggesting a direct interaction between these proteins. (D) BEAF32/CP190 interactions are mediated by CP190-C. A mix of BEAF32/CP190-C was incubated and analyzed by PAGE/Western blotting. Both BEAF32 and CP190-C remain bound to an anti-CP190 column, but not to the control anti-IgG column. (E) A mix of BEAF32, Chromator, and CP190 was incubated and analyzed by PAGE/Western blotting. The three proteins are bound to an anti-Chromator column, but not to the control anti-IgG column. (F) S2 nuclear extracts were incubated in an anti-Chromator or anti-IgG column and analyzed by PAGE/Western blotting. Both BEAF32 and Chromator remain bound to the anti-Chromator column, suggesting that these proteins interact in vivo. (G) S2 nuclear extracts were incubated in an anti-CP190 or anti-IgG column and analyzed by PAGE/Western blotting. Consistent with previous results, BEAF32, CP190 and Chromator remain bound to the anti-CP190 column, suggesting that these proteins are part of the same complex in vivo.

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