Figure 1. The PS1 L435F FAD mutant strongly impairs APP processing while generating Aβ43.
Pooled clones (A–D) and single‐cell clones (E–H) of HEK293/sw cells untransfected or stably transfected with the indicated WT and mutant PS1 constructs were analyzed for γ‐secretase expression and APP processing.
- PS1, PS2, and NCT were analyzed in cell lysates by immunoblotting using antibodies PS1N (PS1), BI‐HF5C (PS2), and N1660 (NCT), respectively.
- Full‐length APP and APP CTFs were analyzed by immunoblotting using antibody 6687.
- Conditioned media were analyzed for secreted APPs by immunoblotting using antibody 22C11 and for total Aβ by combined immunoprecipitation/immunoblotting using antibodies 3552/2D8.
- Conditioned media were analyzed for individual Aβ species on Tris‐Bicine‐Urea SDS‐PAGE by combined immunoprecipitation/immunoblotting of Aβ using antibodies 3552/2D8. Pooled clones of HEK293/sw cells stably transfected with PS1 L166P were used as reference. Note that more sample was loaded for the PS1 L435F mutant to facilitate analysis.
- PS1 expression levels were analyzed in cell lysates of HEK293/sw cells stably expressing PS1 WT or PS1 L435F by immunoblotting using antibodies PS1NT and 5E12, respectively.
- Conditioned media were analyzed by ELISA specific for Aβ40, Aβ42, and Aβ43. Data represent mean ± s.e.m. (n = 6). Absolute levels and Aβ ratios are shown.
- ecreted Aβ was analyzed as in (D). To verify individual Aβ species, Aβ standards were co‐migrated.
- Total Aβ in conditioned media was analyzed by MALDI‐TOF MS following immunoprecipitation with antibody 4G8. Observed (Aβ42, 4513.6; Aβ43, 4615.3) and predicted molecular masses (Aβ42, 4514.1; Aβ43, 4615.2) were in good agreement.