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Fig. 6. Genetic and chemical inhibition of NRADD processing by -secretase. (A) Wild-type (+/+) and PS1,2 double knockout (-/-) cells were transfected with the three NRGV constructs. Luciferase assays were performed as follows. Cells were co-transfected with GV constructs, Gal4 luciferase reporter and renilla luciferase-TK reporter to normalize for transfection efficiency. The -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. (B) Cell lysates were analysed by western blot with anti-NRADD antibody. (C) N2a cells were transfected with NR45GV and incubated with various concentrations of the -secretase inhibitors DAPT or compound E. Luciferase activities were normalized to the activation obtained without inhibitor. Expression of NR45GV was monitored by western blot of the cell lysates (bottom). All error bars represent 95% confidence limit.

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