Figure 1.

Inhibition of Rab5 increases Aβ peptides. (a) To verify the role of endocytosis in the vardenfil (VDF)-stimulated Aβ₄₂ release, N2a cells were pretreated with 25 μM PITSTOP2 for 10 min and then incubated for 1 h with 100 μM VDF or an equal volume of vehicle (DMSO). At the end of the incubation period, conditioned media were subjected to specific Aβ₄₂ ELISA. Graphed data show mean ± SEM of four independent experiments (*P < 0.05; ***P < 0.0001 vs the vehicle-treated group). (b) N2a cells treated with 100 μM VDF or vehicle for 1 h were processed for total protein extraction. Protein extracts were then subjected to Aβ₄₀ ELISA. Graphed data show mean ± SEM of four independent experiments (**P < 0.01 vs vehicle-treated group). (c) Where indicated, N2a cells were transfected with HA-tagged Arf6 (WT), HA-Arf6 Q67L (CA), HA-Arf6 T27N (DN), or with mCherry-Rab5 (WT), mCherry-Rab5 Q79L (CA), and mCherry-Rab5 S34N (DN). After 24 h, conditioned media were subjected to specific Aβ₄₂ ELISA, while cells were processed for total protein extraction followed by immunoblot analysis performed with anti-Rab5 and anti-HA antibodies. The β-actin signal represents the internal loading control. The image shows cropped blots and is representative of three independent experiments; graphed data show mean ± SEM (*P < 0.05; **P < 0.005 vs control group). (d) N2a cells were transfected with Rab5 siRNA or non-targeting siRNA (CNT siRNA). After 48 h, media were changed and collected 1 h later for Aβ₄₀ and Aβ₄₂ ELISA. At the same time, cells were processed for Rab5 immunoblotting. The β-actin signal represents the internal loading control. The image shows cropped blots and is representative of three independent experiments; graphed data show mean ± SEM. (*P < 0.05 vs corresponding control).