FIG. 5.

Deletion of the RAM domain from N^ic constructs results in loss of CBF1-responsive reporter activity and abolishes binding. (A) HeLa cells were transiently transfected with the indicated N^ic expression vector (0.8 μg), 8x-CBF1-luc plasmid (0.4 μg), and RL-TK plasmid (0.4 μg). At 48 h posttransfection, luciferase values were determined for each construct and normalized as described in Materials and Methods (given as relative luciferase units). Shown are the results from one experiment done in triplicate which are representative of multiple assays performed with each deletion construct. (B) N^icΔR mutant constructs do not physically associate with GST-CBF1 fusion polypeptide. Cellular lysates were made from stable RKE cell lines expressing the indicated N^ic construct. Lysates were incubated with either GST-CBF1 beads (lanes P) or GST beads (lanes C). N^ic deletion constructs were detected by Western blotting against the Myc epitope with 9E10. An equivalent amount of each lysate was immunoprecipitated with anti-Notch1 polyclonal antiserum 925 and detected by Western blotting against the Myc epitope (shown in Fig. 2C). Molecular mass markers are shown to the left.