Fig. 4

Aβ−40 and Aβ−42 protein expression study. Aβ−40 and Aβ−42 protein expression levels in cell media of each group. WT and indicated mutants were co-expressed with the APP Swedish mutant in HEK239 cells and the conditioned media were harvested 48 h post-transfection for ELISA-determination of Aβ−40 and Aβ−42. Cell lysates were subjected to Western blot for APP, PS1, PS2, and β-actin as an internal standard. A Western blotting of cell lysates and quantification of Aβ42, Aβ40, and ratios of Aβ42 to Aβ40 relative to PS1 WT in conditioned medium of cells expressing PS1 WT and PS1 Q56R, L174P, S289P, R377M, and Y466C mutants. The Aβ levels were normalized to the total protein levels in PSEN1 WT-expressing cells. Quantifications of the full-length PS1 in the corresponding cell lysates relative to PS1 WT were also provided. The PS1 levels were normalized to the β-actin levels. B Western blotting of cell lysates and quantification of Aβ42, Aβ40, and ratios of Aβ42 to Aβ40 relative to PS2 WT in conditioned medium of cells expressing PS2 WT and PS2 V214L, M239T mutants. The Aβ levels were normalized to the total protein levels in PSEN2 WT-expressing cells. C Western blotting of cell lysates and quantification of Aβ42, Aβ40, and ratios of Aβ42 to Aβ40 relative to APPSwe in conditioned medium of cells expressing Mock (empty vector transfected), APPSwe, D197N (APPSwe combined with D197N variant), and D252V (APPSwe combined with D252V variant) mutants. The Aβ levels were normalized to the total protein levels in APPSwe-expressing cells. All Aβ level normalizations were performed relative to the total protein levels, which may reflect the number of cells. This experiment was performed three times with reproducible similar results