Localization of APP-N2C50 heterodimers was modulated by OSBP1. A, Diagram showing the BiFC method. APP and Notch constructs with a C-terminal deletion (N2C50) were fused with the amino or carboxy domains of YFP. Binding of target constructs brings the two halves of YFP together and produces fluorescence. B, Cells were transfected with APPYC and N2C50YN with or without OSBP1 and, after 24 h, treated with 1 μM 25OH or vehicle for another 24 h. Detection of the BiFC fluorescence was carried out as described and cells were examined under 63× magnification. C, Panels (a) and (b) show fluorescence images of cells expressing P450 2C2/CFP (ER CFP) and 1,4-galactosyltransferase/DsRed2 (Golgi DsRed) markers, acquired 24 h after transfection; panels (c) and (d) show localization of OSBP1 in controls and cells treated for 24 h with 1 μM 25OH, respectively.