Figure 8.

TFEB expression reduces steady-state cell-surface and intracellular APP levels without altering its endocytosis. A, B, Immunoblot (A) and quantitation (B) of cell-surface and internalized APP in N2a-APP695 cells transfected with TFEB or vector control, after cell-surface APP was labeled by biotinylation at 4°C (to prevent endocytosis) followed by rewarming (to 37°C) to stimulate its internalization by endocytosis (for 10 min). Cell-surface APP was assessed in cells maintained at 4°C. To assess internalized APP, cells were treated with 2-mercaptoethanesulfonic acid to remove cell-surface biotinylated APP molecules, followed by streptavidin capture and immunoblotting (see Materials and Methods). Inset shows ratio of internalized to surface APP; *p < 0.05. C–E, Immunoblot (C) and quantitation (D) of intracellular APP as a fraction of cell-surface APP to demonstrate kinetics of intracellular Aβ in cells transfected as in A and kinetics of uptake of biotinylated transferrin [immunoblot (C) with quantitation (E)] in cells treated as in A. N = 3 per group. No statistically significant differences were observed by two-way ANOVA.