Figure 1From: Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminantsAPP 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-APP1–17 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.Back to article page