FIG 5.

NURF directly interacts with Ctcf and the cohesin subunit SA2. (A) Results of a representative in vivo co-IP experiment from total ESC extracts using antibodies to Ctcf, Oct1, Sp1, and Nfatc1 and a control IgG. Bptf co-IP with Ctcf pulldown was detected by Western blotting with an antibody to Bptf. Ctcf, Oct1, Sp1, and Nfatc1 IP was confirmed by Western blotting with the antibodies used for pulldown. (B) Results of a representative in vivo co-IP experiment from total ESC extracts using antibodies to Ctcf, Smc1a, Smc3, Rad21, and SA2 and a control IgG. Bptf co-IP with Ctcf and SA2 pulldown was detected by Western blotting with an antibody to Bptf. Ctcf, Smc1a, Smc3, Rad21, and SA2 IP was confirmed by Western blotting with the antibodies used for pulldown. (C) Results of a representative in vivo co-IP experiment from total ESC extracts using an antibody to Bptf and a control IgG. Co-IP of Ctcf and SA2 were detected by Western blotting with antibodies to Ctcf and SA2, respectively. Bptf IP was confirmed by Western blotting using same antibody for pulldown. (D) Results of a representative in vitro pulldown experiment of purified recombinant human NURF complex (containing a FLAG-Bptf subunit) by resin-bound human GST-CTCF, GST fusions to the human cohesin subunits SMC1a, SMC3, RAD21, and SA2, or a GST control. Pulldown of FLAG-BPTF was detected by Western blotting with an antibody to FLAG. (E) Representative in vitro pulldown of recombinant human NURF complex (containing a FLAG-BPTF subunit) by resin-bound GST N-terminal (NT-CTCF), zinc finger (ZF-CTCF), and C-terminal (CT-CTCF) Ctcf fusions. Pulldown of FLAG-BPTF was detected by Western blotting with an antibody to FLAG. (F) Results of a representative pulldown of recombinant human NURF complex (containing a FLAG-BPTF subunit) by resin-bound GST fusions with the N-terminal (NT-SA2), center (CN-SA2), and C-terminal (CT-SA2) regions of SA2. FLAG-BPTF was detected by Western blotting with an antibody to FLAG.