OSBP1 overexpression inhibited β-secretase cleaved APP-CTF (CTFβ) and decreased sAPPβ, Aβ40, and Aβ42 secreted levels, without affecting total or cell surface steady-state levels of full-length APP. A, H4-APP cells were transfected with OSBP1. After 48 h, medium was removed and replaced with fresh optiMEM. Following 3 h incubation, media were collected and concentrated using Vivaspin columns. Proteins from lysates and media were separated by SDS-PAGE and media samples were immunoblotted using the 192 wt antibody, against the β-cleaved epitope of APP (top panel); lysates were blotted with anti c-myc antibody to confirm OSBP1 expression (middle panel) and with 6E10 antibody, which recognizes APP-CTFβ (lower panel). Densitometric analysis is shown on the right. B, Secreted Aβ40 and Aβ42 levels in 24 h conditioned media from mock transfected and OSBP1-overexpressing HEK-APPNFEV cells were measured by ELISA. C, HEK-APPNFEV cells were transfected with OSBP1 cDNA as described in Methods. After 48 h incubation, cell monolayers were biotinylated prior to lysis. Total and cell-surface biotinylated proteins were separated and analyzed by Western blotting using antibodies to OSBP1, APP and actin as loading control. *p < 0.05 and **p < 0.01 by Student's t test.