Coimmunoprecipitation of EAF2 and ELL.

(A) Specificity of EAF2 and EAF1 monoclonal antibodies. 293 cells were transfected with FLAG-EAF1 or FLAG-EAF2 constructs. The cell lysates were immunoprecipitated with each of the monoclonal antibodies and blotted with the FLAG antibody. The 1E6 antibody precipitated FLAG-EAF1 but not FLAG-EAF2. In contrast, the 7A4, 1F11, and 5F6 monoclonal antibodies precipitated FLAG-EAF2 but not FLAG-EAF1. The 6C6 monoclonal antibody precipitated FLAG- EAF1 and FLAG-EAF2 equivalently, indicating that this antibody recognizes an epitope that is shared by both proteins. (B) Endogenous EAF2 is in a complex with endogenous ELL in untransfected cells. 293 cell extracts were immunoprecipitated with an isotype-control antibody or with the 3 monoclonal antibodies specific for EAF2. Using an affinity-purified polyclonal ELL antibody, endogenous ELL was detected in the lysates precipitated by the EAF2 antibodies but not in the lysates precipitated by the isotype control. (C) Endogenous EAF2 interacts with transfected ELL2. 293 cells were transfected with FLAG-ELL, FLAG-ELL2, or FLAG-ENL. Expression of these constructs was demonstrated by Western blot analysis of cell lysates with the FLAG antibody (left panel). Cell extracts were immunoprecipitated with the FLAG antibody. Endogenous EAF2 coprecipitated with FLAG-ELL and FLAG-ELL2, as detected by Western blot analysis using the 1F11 anti-EAF2 antibody (right panel). However, EAF2 did not coprecipitate with FLAG-ENL. Endogenous EAF2 migrates at approximately 42 kDa.