FIG. 2.

In vivo interaction of ETO with HDAC1 and mapping of the ETO CoR-binding region. (a) Mapping of the CoR interaction domain in ETO. In vitro-translated ETO or mutant ETOs were precipitated with GST or a GST-SMRT RD fusion protein. ETOΔC contains amino acids 1 to 416, ETOΔZnF lacks amino acids 488 to 525, and ETO-C488S and -C508S represent point mutations of the indicated amino acids. All of the ETO proteins were hemogglutinin fusions, except ΔC, which was a Gal4 fusion. (b) In vivo interactions of ETO and endogenous N-CoR. Coimmunoprecipitations of N-CoR and ETO or ETOΔC. Immunoprecipitates with anti-N-CoR antibodies or control immunoglobulin G (IgG) were obtained from 293T cells transfected with a Flag-ETO or Flag-ETOΔC expression vector and blotted with an anti-Flag antibody. The input lane contains 2% of the total. (c) In vivo association of ETO and HDAC. HDAC activity of the indicated immunoprecipitates from 293T cells transfected with the indicated expression vectors. (d) In vivo association of ETO and HDAC. Coimmunoprecipitation of ETO and HDAC1. Immunoprecipitates with an anti-Flag antibody or control immunoglobulin G were obtained from 293T cells transfected with a Flag-ETO or Flag-ETOΔC expression vector and blotted with an anti-HDAC1 antibody. The input lane contains 2% of the total. (e) Lack of direct interaction between HDAC1 and ETO in vitro. In vitro-translated HDAC1 was precipitated with GST or GST-ETO. IP, immunoprecipitation; dpm, disintegrations per minute.