PHD/Bromo region interacts with ASB2 and mediates MLL ubiquitination. (A) Schematic diagram of the structure of wild-type MLL. The CxxC and CxxC-PHD/Bromo fragment used in immunoprecipitation are shown with the first and last MLL amino acid retained in the constructs indicated. (B) Myc tagged CxxC or CxxC-PHD/Bromo (CxxC-P/B) was coexpressed in 293 cells with HA tagged ASB2. Cells were treated with MG132 for 6 hours. Immunoprecipitation of CxxC or CxxC-PHD/Bromo followed by Western blotting shows that ASB2 and endogenous EloB and EloC specifically interact with CxxC-PHD/Bromo. (C) NB4 cells were treated with ATRA for 48 hours and with MG132 for 16 hours. MLL was immunoprecipitated with an anti-MLL antibody, and Western blotting using an anti-ASB2 antibody shows that endogenous ASB2 coprecipitates with MLL. (D) Flag tagged MLL or MLL-AF9 was coexpressed in 293 cells with HA tagged ASB2. After MG132 treatment, anti-Flag immunoprecipitation was performed followed by Western blotting, showing that ASB2 interacts with MLL but not with MLL-AF9. (E) Myc tagged CxxC or CxxC-PHD/Bromo and Flag tagged ubiquitin were coexpressed in 293 cells. Immunoprecipitation of CxxC or CxxC-PHD/Bromo was performed after MG132 treatment. Western blotting shows that CxxC-PHD/Bromo is conjugated with ubiquitin.