Figure 5 LRP1 induces a shift in the subcellular localization of BACE1 from early to late endosomes in both HEK293 and primary neurons, likely promoting lysosomal degradation. A, HEK293 cells transfected with BACE1 plus LRP-L4 (B+L) or BACE1 only (B only) were analyzed by triple-immunofluorescence staining with anti-EEA1/anti-rab7a (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-EEA1/anti-rab7a (green) and 1D4 (red) antibodies. Note that colocalization of HA, 1D4, and EEA1/rab7a immunoreactive signals was observed in cells expressing BACE1 and LRP-L4, whereas colocalization of signals for 1D4 and EEA1, but not rab7a, was observed in cells expressing BACE1 only. Scale bars, 20 μm. B, HEK293 cells transfected with BACE1 plus LRP-L4 or BACE1 only were analyzed by triple-immunofluorescence staining with anti-β-COP/anti-γ1-adaptin (green), anti-HA (red), and 1D4 (blue) antibodies, or double-immunofluorescence staining with anti-β-COP/anti-γ1- (continued in page 11). adaptin (green) and 1D4 (red) antibodies, as in A. C, Primary neurons grown on coverslips were infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses. Cells were analyzed as in A, except that goat anti-EEA1 and rabbit anti-HA antibodies were used. Neurons coexpressing BACE1 and LRP-L4 exhibited overlapping 1D4, HA, and EEA1/rab7a immunoreactive signals in soma and neurites, whereas neurons expressing only BACE1 exhibiting overlapping signals of 1D4 and EEA1, but not rab7a. Scale bars, 20 μm. D, Primary neurons infected with BACE1 plus LRP-L4 adenoviruses or BACE1 plus LacZ adenoviruses were analyzed by triple- or double-immunofluorescence analysis performed with anti-β-COP/anti-γ1-adaptin, anti-HA, and 1D4 antibodies, as in B. Scale bars, 20 μm. E, HEK293 cells transfected with BACE1 plus LRP-L4 were subjected to cycloheximide (CHX) chase experiments, in which cells were coincubated with or without chloroquine (CQ; 50 μM). After 12 h, cells were lysed and analyzed by Western blotting. Relative levels of BACE1 and LRP-L4 were quantified and graphed (n = 3, *p < 0.05).