Composite CTCF Binding Sites Figure 5. CTCF and TR synergize in enhancer blocking. Cells transfected with episomes were incubated for 48 h in the absence or presence of T3. ESRRA = abbr. ERR. (A) 293T cells were transfected with the indicated episomes, a TRb expression vector, and with either the CTCF expression vector (CTCF) or an empty vector (vector). (B) A 293T cell clone containing a stably integrated vector with Tet-inducible expression of shRNA against CTCF was transfected with the indicated reporter episome and the TRb expression vector. The cells were incubated with tetracycline for 0, 24 or 48 h. (C) Indicated cell lines expressing either a low amount of or no TRb were transfected with reporter episomes and either empty vector (vector) or TRb- expression (TRb). (D) EMSA experiments were performed using E.coli expressed GST, GST-CTCF-ZF and GST-TR and radioactively labeled F1/F2 probe. For competition experiments, non-labeled probes (N) were used in amounts as indicated. (E) Either the mutated F1 (F1mutF2) or the mutated F2 (F1F2mut) were tested in N2a cells in the presence of TR-expression vector. doi:10.1371/journal.pone.0010119.g005 PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10119