C-terminal Notch activating mutations signal by alternative mechanisms. (A) Schematic diagram showing the intracellular domain of Drosophila WT Notch and mutant constructs used, PPxF, ΔPEST, and ANK. NB all constructs have intact ECD (not shown). (B) Overactivation of the mutant Notch constructs and WT+Dx, analyzed by NRE-luciferase assay in S2 cells. ***P < 0.001 compared to WT Notch. (C) Activation of WT Notch and mutant constructs with and without treatment of cells with BB-94, MβCD, or ML-SI1. *, **, and *** indicate P < 0.05, 0.01, and 0.001 by two-tailed t test, respectively for comparisons indicated on the graph. Error bars represent SEM. Sample sizes are indicated in figure. (D) Time course of colocalization between Notch^ECD antibody uptake and Cav-1-mRFP. Notch^ECD antibody was labeled for 15 min (pulse), then chased for 0, 10, 30, and 60 min, and the percentage of Notch^ECD on Cav1-mRFP positive spots was scored with WT Notch, PPxF, or ANK expressing cells. Data from 3 experimental repeats, with 50–100 puncta scored per repeat, error bars SEM.