Inhibition of Notch family members in vitro by MAML1-3 dominant negative mutants. Dominant negative mutants were generated from homologous regions in the N-terminal Notch-binding domain of MAML1, MAML2, and MAML3 (mMAML1-3). (A) Transient transfection of U2OS cells with a CSL reporter plasmid and combinations of plasmids encoding the constitutively active intracellular domain of Notch family members (ICN1-4) and mMAML1-3. Firefly luciferase activity was normalized to Renilla luciferase activity. The amounts of each plasmid used are indicated in “Materials and methods.” Relative activity is shown on the y-axis. Results are shown as the mean ± SD of triplicate samples. (B) 293 T cells were transfected with plasmids encoding ICN1 and DNMAML-GFP fusion constructs versus GFP alone, in the presence or absence of myc-tagged CSL. The amounts of each plasmid used are reported in “Materials and methods.” Immunoprecipitates prepared with anti-myc antibody were analyzed on Western blots stained with anti-myc, anti-Notch, or anti-GFP antibodies. The lower molecular weight bands revealed by anti-GFP antibodies in the DNMAML-GFP transfected samples are protein degradation products. (C) Schematic map of the MSCV-based retroviral constructs used in this study.