Figure 1

APP processing. (A) Schematic representation of APP processing. APP undergoes two alternative endoproteolytic pathways. In the amyloidogenic pathway, APP is first cleaved by β-secretase (β) to enable the production of the membrane bound C99 fragment. C99 is then cleaved in its intramembranous domain by γ-secretase (γ) to release Aβ. In the non-amyloidogenic pathway, APP is cleaved by α-secretase (α) to generate sAPPα and the membrane bound C83 fragment. Immunogenic regions for the indicated anti-APP antibodies are shown. (B) HEK293 cells stably transfected with Swedish human APP₆₉₅ cDNA [16] were incubated in the absence (Control) or presence of γ-secretase inhibitor (L-685,458, Calbiochem, 1 μM for 18 hrs). APP full-length (APP) and C-terminal fragments (C99 and C83) were analyzed by western blotting (WB) as described before [16], using antibodies directed against APP N-terminal domain (22C11, Chemicon) and APP C-terminal domain (R1, provided by Dr. P.D. Mehta, see Ref. [16]), respectively. Secreted sAPPα and Aβ were analyzed by WB using anti-APP_1–17 antibody (6E10, Signet). Wild type and APP-transfected HEK293 cells were grown in DMEM plus 10% FBS, penicillin and streptomycin in 5% CO₂ at 37°C. APP-transfected HEK293 cells were selected and maintained in 5 μg/ml puromycin.