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. 2012 May 14;109(22):8534–8539. doi: 10.1073/pnas.1202691109

Fig. 2.

Fig. 2.

Potent NCT-specific Fab2 inhibits γ-secretase activity in vitro. (A) Schematic illustration of full-length human NCT and truncated NCT variants. SP, signal peptide; TM, transmembrane; TTR, transthyretin; 6HIS, His tag. (B) Immunoprecipitation of ECD (Upper) or 716 (Lower) from HEK293 cells with Fab12 (lanes 3), Mab5226A (lanes 4), or Fab2 (lanes 5). (C) Fab2 immunoprecipitation of secreted NCT fragments in medium of GnTI− cells (Top) in the absence (Middle) or presence (Bottom) of 0.1% Nonidet P-40. “con Fab” is a negative control Fab raised gainst an unrelated target. (D) SPR sensorgrams of Fab2 (ECD/Fab2 and 716/Fab2) or Fab12 (ECD/Fab12 and 716/Fab12) binding to immobilized ECD (Left and Center Left) or 716 (Right and Center Right); kinetic values are shown in the table. (E) Effects of Fabs on γ-secretase activity. Purified γ-secretase from HEK293S TAP-ANPP cells (Left). In vitro γ-secretase activity assays using purified γ-secretase preincubated with different Fabs; production of Aβ (Center) and NICD (Right) were monitored by ELISA. Data were represented as mean ± SEM, n = 3.