Mapping the interaction between ETO and Bcl-6 in vitro and in vivo. (A) Protein-affinity chromatography was performed to map the interacting domains of ETO and Bcl-6. Schematic representations of the bacterially expressed GST-ETO and in vitro–transcribed/translated Bcl-6 constructs are shown. Results of the interactions between the different fragments after GST pull-down is shown by fluorography of the 35S-methionine–labeled Bcl-6 constructs. (B) Coimmunoprecipitation was performed in 293T cells cotransfected with myc-tagged Bcl-6 and the GAL4-ETO fusion constructs shown in the schematic representation. The upper panels show 2% input from immunoblots performed with GAL4 polyclonal antibodies and myc-tagged monoclonal antibodies. The bottom panels show immunoblots resulting after immunoprecipitation with myc-tagged mouse monoclonal antibody for Bcl-6 (B6) or mouse control IgG (Ig). The analysis is separated into 4 boxes to show separately the area from the N- to the C-terminus of the ETO protein interacting or not interacting with Bcl-6. (C) Myc-tag Bcl-6 (Bcl-6) or GAL4–Bcl-6 deleted for the zinc finger (Bcl-6ΔZincF) was cotransfected with HA-ETO in 293T cells. Coimmunoprecipitation was performed with the ETO N-terminal polyclonal antibody or its IgG control. Proteins (2% input or immunoprecipitate) were run on 15% SDS-PAGE and immunoblotted with the Bcl-6 mouse monoclonal antibody and the HA antibody.
