Characterization of NHR4/N-CoR and AML1-ETO/SON binding and effects on gene repression. (A) Structures of AML1-ETO and the NHR2-4 constructs used in the co-IP experiments, with location of N-CoR binding sites indicated. (B) Endogenous N-CoR interacts with WT, but not W692A, NHR2-4. Cell lysates were immunoprecipitated with N-CoR or isotype control (ctrl) antibodies, and N-CoR and NHR2-4 were detected with N-CoR and Flag antibodies, respectively. Tubulin serves as a loading control. (C) SON interacts with both WT and W692A NHR2-4. Cell lysates were immunoprecipitated with HA antibody, and SON and NHR2-4 were detected using HA and Flag antibodies, respectively. (D) SON interacts with full-length AML1-ETO (AE), but not when the NHR3 domain is deleted (ΔNHR3) or when amino acids 629-634 ‘TERAKM’ in NHR3 are mutated to ‘AAAAAA’ (mutNHR3). Immunoprecipitation is as performed in (C). (E) Endogenous N-CoR retains interaction with NHR2-4 containing mutNHR3 alanine mutations. Immunoprecipitation performed as in (B). (F) W692A decreases ability of AML1-ETO to repress a luciferase reporter; mutNHR3 does not. 293T cells were cotransfected with firefly luciferase reporter containing tandem AML1 binding sites upstream of the basal TK promoter, Renilla control luciferase and either empty vector (control) or indicated AML1-ETO construct. Firefly luciferase expression was normalized to Renilla expression and control was set to 1. Equal expression of AML1-ETO constructs was confirmed by immunoblot using HA antibody (data not shown). Data show averages and standard deviations of 3 independent experiments, each performed in duplicate. EV, empty vector; WCL, whole cell lysate.