Fig. 7. -Secretase activity is not influenced by the glycosylation status of nicastrin. (A) Cell-free -secretase assay using membranes from wild-type (WT) MEFs treated with MNJ. C–, negative control (substrate only); C+, positive control (solubilized -secretase from Hela cells); –s: solubilized -secretase from WT MEFs without substrate added. +s: substrate added. (B) Western blot analysis of the membranes used in panel A showing the absence of glycosylation maturation of nicastrin after MNJ treatment. (C) Cell-based -secretase assay. HEK cells stably expressing APP/Sw were transiently transfected with mNotchE or NICD and treated with kifunensine (1 µg/ml), a mannosidase I inhibitor resulting in inhibition of mature glycosylation of nicastrin. Aß and NICD generation was analyzed as described. Note that inhibition of glycosylation of nicastrin does not inhibit the production of Aß nor NICD. (D) Inhibition of glycosylation has no effect on surface expression of nicastrin. HEK293 cells were treated with kifunensine (1 µg/ml) and surface biotinylated after 72 hours kifunensine treatment. Biotinylated proteins were precipitated using streptavidin beads and immunoblotted with B59.2 (-nicastrin). Western blot analysis of total cell extracts is shown as a control. Nicastrin is labeled in both treated and untreated cells, demonstrating that immature glycosylated nicastrin can reach the cell surface under these conditions.