Fig. 6

Sorting nexin-4 (SNX4) shifts β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) away from the degradation pathway. a Gradient fractionation was performed on SH-SY5Y cells cotransfected with BACE1 and either mock or SNX4 constructs. In mock-transfected cells, strong BACE1 signals appeared in fractions 6 and 7, which overlap with Rab7, a late endosome marker. BACE1 appeared primarily in fractions 3–5, which overlap more with Rab11, a recycling endosome marker. E.E., early endosome; R.E., recycling endosome; L.E., late endosome. b Gradient fractionation was performed on SH-SY5Y cells cotransfected with BACE1 and either scrambled small interfering RNA (siCTL) or small interfering RNA mixture (siSNX4). In siCTL-transfected cells, strong BACE1 signals appeared in fractions 6–10, which overlap with Rab7 and Rab11. BACE1 appeared primarily in fractions 7–10, which overlap more with Rab7. c Immunocytochemistry was performed using an anti-BACE1 antibody (red) and anti-Rab11 antibody (green) in SH-SY5Y cells. Arrows indicate BACE1 localization to the cell membrane. Scale bar = 20 μm. d HEK293 cells expressing BACE1 were transfected with mock or SNX4 constructs and cotreated with cycloheximide, a protein synthesis inhibitor. BACE1 decreased very rapidly after 1 h in mock-transfected cells but decreased slowly in SNX4-transfected cells. ****p < 0.001. EEA1, Early endosome antigen 1