FIGURE 8.

Syvn facilitates the trafficking of mature NCT and the assembly of the γ-secretase complex. A, Western blot analysis of cell surface proteins and lysates prepared from WT and Syvn^−/− fibroblasts with the indicated antibodies. Cell surface proteins were biotinylated (Biotin+) and isolated as described under “Experimental Procedures.” Non-biotinylated cells (Biotin-) were used as a negative control. The same volume of eluted sample (Cell Surface) and the same amount of protein (Lysate) were loaded in each lane. Integrin β1 served as a loading control (left panel). Quantitation of cell-surface NCT levels from four independent experiments is shown in the left panel (right graph). Values are expressed as mean ± S.D. *, p < 0.01 (two-tailed Student's t test). B, Blue Native PAGE (BN-PAGE) and SDS-PAGE analysis of lysates prepared from WT and Syvn^−/− fibroblasts as described under “Experimental Procedures” and blotted with anti-NCT (left panel), anti-Aph1 (middle panel), and anti-PS1 antibodies (right panel).