Figure 2.

Analysis of secretion deficient mutants in HEK293 cells. (A) Western Blot analysis of APP secretion deficient mutants. HEK293 cells were transiently transfected with APPwt and mutants APPΔ622 and APP-D8. Cell lysates were analyzed with antibody 22C11 or W02 to detect full length APP via Western Blot. Medium samples of APP were analyzed with antibody 22C11 to detect sAPP_total, antibody W02 to detect sAPPα and an sAPPβ specific antibody. Note, the W02 epitope is deleted in APPΔ622. β-actin antibody served as a loading control. (B) Quantification of data shown in panel (A). One-way ANOVA followed by Tukey’s post hoc analyses (* p < 0.05; ** p < 0.001; *** p < 0.0001, n = 5 ± SEM). (C) Cell surface biotinylation of APP secretion deficient mutants. HEK293 cells were transfected with the indicated constructs. Direct load of cell lysates is documented in the left panel together with the β-actin loading control. In the right panel, APP cell surface levels after streptavidin immunoprecipitation and Western Blot detection with antibody c-myc is shown and β-actin as a negative control for intracellular proteins at the cell surface.