Fig. 1. DAPT blocks Notch endoproteolysis and inhibits a zfPS1-dependent γ-secretase and S3 protease activity in HEK293 cells. (A) HEK293 cells stably transfected with the NotchΔE cDNA (Schroeter et al., 1998) were treated with or without 1 µM DAPT for 4 h. Cell lysates were analyzed for NotchΔE (NΔE; uncleaved form of Notch) and NICD (cleaved form of Notch) by immunoblotting using antibody 9E10 to the myc tag at the C-terminus of these Notch variants. (B) Panels 1–3: cell lysates from HEK293/sw cells stably transfected with the indicated zfPS1 cDNAs were analyzed by immunoblotting with antibody zfPS1_loop, 3027 to human PS1 or 3711 to human PS2. Note that endogenous human PS1 and PS2 are replaced by zfPS1 variants. Replacement of endogenous PSs by overexpressed PS variants is an important indication for incorporation of the ectopic PS into a biologically functional PS complex (Thinakaran et al., 1997). Panel 4: cell lysates were analyzed for APP CTFs by immunoblotting with antibody 6687. Panel 5: conditioned media were analyzed for total Aβ (Aβ40 and Aβ42) species by immunoprecipitation/immunoblotting with antibodies 3926/6E10. Immunoprecipitates were separated on a Tris–bicine–urea gel that allows the identification of Aβ40 and Aβ42 (Wiltfang et al., 1997). Note that expression of wt zfPS1 not only allows robust Aβ (Aβ40 and Aβ42) production, but also leads to increased production of Aβ42 (Leimer et al., 1999). (C) HEK293/sw cells expressing either wt zfPS1 or mutant zfPS1 D374N were stably transfected with the NotchΔE cDNA (Schroeter et al., 1998) and cell lysates were analyzed for NotchΔE (NΔE) and NICD as in (A). (D) HEK293/sw cells stably expressing wt zfPS1 were treated with or without 1 µM DAPT for 4 h and analyzed for APP CTFs as in (B) and for Aβ by direct immunoblotting with antibody 3926. (E) HEK293/sw cells stably expressing wt zfPS1 were treated with increasing amounts of DAPT for 4 h and cell lysates were analyzed for NotchΔE (NΔE) and NICD as in (A).