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Fig. 5. Truncated NRADD is a {gamma}-secretase substrate. (A) The GV constructs. The amino acid numbering refers to wild-type NRADD. The two glycosylation sites are indicated in the extracellular domain (ECD) of FL-NRGV. (B) Expression of the GV constructs in 293 cells. The western blot was probed with anti-NRADD antibody. (C) Luciferase assay in N2a cells. Cells were co-transfected with GV constructs, Gal4 luciferase reporter and renilla luciferase-TK reporter to normalize for transfection efficiency. The {gamma}-secretase inhibitor L-685,458 (10 µM) was added 5 hours after transfection. Cells were lysed 48 hours after transfection. Dual luciferase assays were performed and the fold induction calculated. Error bars represent 95% confidence limit. (D) The same lysates were analysed by western blot either before or after N-glycanase digestion. End., endogenous NRADD; Exo., transfected NRADD.





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