Figure 4

Interaction of BCL11A-XL with BCL6, with itself, and with other BCL11A isoforms. (A) XL and L, but not S, associate with BCL6. Top, in vitro translated 35S-labeled BCL11A isoforms were incubated with cell lysates of HEK293 cells transiently transfected with FLAG-BCL6. Complexes were immunoprecipitated with anti-FLAG, separated on SDS-PAGE, and then visualized by fluorography. Bottom HEK293 cells were transiently cotransfected with FLAG-BCL6 and HA-BCL11A isoform constructs. 48 hrs post-transfection, cell lysates were prepared, immunoprecipitates were prepared with anti-FLAG and products resolved as above were detected by anti-HA Western blotting. (B) Identification of endogenous BCL11A-XL and BCL6 heteromeric complexes in B-cell lines. Whole cell lysates from the Burkitt's lymphoma cell line, Raji, were immunoprecipitated with anti-BCL6 polyclonal antisera, resolved on SDS-PAGE and blotted for BCL11A-XL. (C) All BCL11A isoforms interact with each other in vitro. Immunoprecipitations were performed using in vitro translated, unlabeled Gal4-DNA-binding domain (Gal4DBD) alone or Gal4DBD-BCL11A isoform fusion proteins, and in vitro translated 35S-labeled HA-BCL11A isoforms, in the presence or the absence of an anti-Gal4-DNA-binding domain antibody (anti-Gal4DBD). The immunoprecipitated products were separated on SDS-PAGE and visualized by fluorography.