Fig. 5. Soluble, catalytically inactive extracellular domain of ADAM12 inhibits DLL1 processing and stimulates Notch signaling in myoblasts. (A) Coomassie-blue-stained SDS-PAGE gel showing the recombinant, soluble, extracellular domain of mouse ADAM12 containing the E349Q mutation in the catalytic site (protein X), expressed in Drosophila S2 cells and purified from culture medium. The molecular size markers (kDa) are shown on the right. (B) Inhibition of DLL1 cleavage by protein X. COS-7 cells transfected to express DLL1 were incubated for 24 hours in the absence or presence of 2 µM protein X (left panel). Cell extracts from DLL1-transfected and empty vector (V)-transfected cells were analyzed by western blotting using antibody against the cytoplasmic domain of DLL1 (right panel). DLL1^FL and DLL1^TMIC are indicated with the arrows, positions of the molecular size markers are on the right. The extent of DLL1 cleavage was calculated as the ratio of band intensities of DLL1^TMIC and DLL1^FL (mean ± s.e.m., n=3). (C) The effect of protein X on Notch signaling. (a) Primary myoblasts were incubated for 1 day in differentiation medium without protein X or with 2 µM protein X, in the absence or presence of 1 µM DAPT (D). The level of active Notch, NICD, was analyzed by western blotting using an epitope-specific antibody, tubulin is a gel-loading control. The amount of NICD was quantified by densitometry and normalized to the amount of tubulin (mean ± s.e.m., n=3). (b) Primary myoblasts or C2C12 cells were transfected with a CBF1-luciferase reporter and pRL-TK vector, 24 hours after transfection cells were transferred to differentiation medium (Day 0) and incubated for additional 24 hours (Day 1), in the absence (gray bars) or presence (black bars) of 2 µM protein X. The relative firefly and Renilla luciferase activities were measured using the Dual-Luciferase reporter assay. Fold of CBF1 activation over the level at Day 0, in the absence of protein X, was calculated. The data represent the means ± s.e.m. from three measurements, the experiment was repeated twice with similar results.