sAPPα and sAPPβ construct were created by introducing the stop codon immediately following the α-secretase cleavage site (HQK^LV) or β-secreatase cleavage site (VKM^DAE) to mouse APP 695 pcDNA (sAPPΔ). The full-length mouse and sAPPΔ cDNAs were subcloned into pLNCX2 vector (Clontech) and stably transfected into rat B103 neuroblastoma cells with Lipofectamine 2000 according to the manufacturer’s instructions. Following selection for G418 resistance, the cells were pooled and assayed without clonal selection. Levels of sAPP secreted into the medium were measured by Western blotting with rabbit polyclonal antibody 63G which recognizes the extracellular domain of APP (Marquez-Sterling et al., 1997). Rabbit polyclonal antibody CT15 that recognizes the C-terminus of APP was used to verify full-length protein in cell lysates (Soriano et al., 2001). Transfected B103 cells were cultured in DMEM plus 10% fetal calf serum but switched to neurobasal medium with B-27 supplement overnight for conditioning and the medium was harvested and stored at −80 °C until use.
