EAF1 interacts with ELL and ELL2.

(A) The 293 cells were transfected with FLAG-ELL (lanes 1, 5, and 9), FLAG-ELL2 (lanes 2, 6, and 10), FLAG-ENL (lanes 3, 7, and 11), or the FLAG-tagged AF4 activation domain (lanes 4, 8, and 12). Expression of these constructs was demonstrated by Western blot analysis of cell lysates with the FLAG antibody (lanes 1-4). Cell extracts were immunoprecipitated with an isotype control antibody (lanes 5-8) or with the EAF1 monoclonal antibody (lanes 9-12). Endogenous EAF1 coprecipitated FLAG-ELL (lane 9) and FLAG-ELL2 (lane 10), as detected by Western blot analysis using the anti-FLAG antibody. However, EAF1 did not associate with FLAG-ENL or FLAG-AF4 (lanes 11 and 12). The lower band in lanes 5 to 12 is immunoglobulin heavy chain. (B) Transfected 293 cell extracts were immunoprecipitated with the anti-FLAG antibody, divided into 2 lanes each, and probed with either an isotype control antibody or with the EAF1 monoclonal antibody. Endogenous EAF1 coprecipitated with FLAG-ELL and FLAG-ELL2 (lanes 5 and 6), but not with FLAG-ENL or FLAG-AF4 (lanes 7 and 8). As an additional control, the immunoprecipitates were also probed with an isotype control antibody. (C) The carboxy-terminus of ELL binds to EAF1. The 293 cells were transfected with FLAG-tagged constructs containing multiple regions of ELL, immunoprecipitated with the FLAG antibody, and probed with the EAF1 antibody. Endogenous EAF1 coprecipitated with ELL amino acids 46 to 621, the region of ELL contributed to the MLL-ELL fusion protein. Coprecipitation of endogenous EAF1 could also be detected with amino acids 376 to 621 and 401 to 621 of ELL, but not with amino acids 46 to 208 or 207 to 411. An arrowhead indicates the EAF1 band.