DAPT and L-685,458 inhibit γ-secretase activity. (a) Cells were treated overnight with Z-LLNle-CHO at calculated ED50 values, 5 μM of DAPT, or 2 μM of L-685,458 before protein samples were prepared. Extracellular calcium was depleted by incubation with 0.53 mM of EDTA for 10 minutes to activate Notch1 before sample preparation for all samples except the negative controls. Protein samples were then subjected to western blot analysis with an antibody (V1744) that specifically recognizes active Notch1 intracellular domain (product of γ-secretase-mediated cleavage). Stronger V1744 signal intensity indicates greater γ-secretase activity. The treatment conditions were (from lane 1 to lane 5): dimethyl sulfoxide (DMSO) vehicle only and without calcium depletion as negative control; DMSO vehicle only and with calcium depletion to activate Notch1 as positive control; Z-LLNle-CHO at concentrations equal to the IC50 of individual cell lines; DAPT at 5 μM; L-685,458 at 2 μM. (b) MCF-7 (top panels) and SKBR3 (bottom panels) cells were transfected with plasmid DNA expressing a Flag-tagged protein that mimics the immediate substrate of γ-secretase, treated overnight with Z-LLNle-CHO at calculated ED50 values, 5 μM of DAPT or 2 μM of L-685,458, and then immunostained with anti-Flag antibody. The appearance of nuclear Flag signal indicates the presence of active γ-secretase. Please note the γ-value of Flag signal was enhanced to visualize weak nuclear or cytomembrane signal. (c) Protein samples were prepared without calcium depletion from MCF-7 and SKBR3 cells that were transfected and treated in the same way as the cells in panel b and were subjected to immunoblotting with anti-Notch1 (V1744) antibody. The treatment conditions were (from lane 1 to lane 4): DMSO vehicle only; DAPT; L-685,458; and Z-LLNle-CHO.