Fig. 4.
Effect of a Ncstn mutation in mature and functional γ-secretase complex formation. (A) Immunoblot analysis of NCSTN, APP-CTF, and PS1-NTF in TCLs of thymus, spleen, lymph nodes, brain, skin, splenic B220+, and CD3+ T cells from NcstnV439G/V439G mice and wild-type littermates. The red arrowhead indicates the mature form of NCSTN; blue arrowhead, the intermediate form of NCSTN. (B) Glycosidase treatments of NCSTN in splenocytes isolated from NcstnV439G/V439G mice or wild-type littermates. Endo H and PNGase F treatments show the sensitivity of the various bands to deglycosylation. The purple arrowhead indicates nonglycosylated NCSTN. (C) TCLs isolated from Ncstn−/− MEFs stably expressing FLAG-tagged wild-type, mutant (V439G) NCSTN, or empty vector (EV) were immunoprecipitated using anti-FLAG M2 agarose beads and immunoblotted with antibodies against FLAG, PS1, and PEN2. The red arrowhead indicates the mature form of NCSTN; blue arrowhead, the intermediate form of NCSTN; black arrowhead, full-length PS1; green arrowhead, PS1-NTF. (D) BN-PAGE analysis of γ-secretase subunits (nicastrin, PS1, PEN2, and APH1) from an equivalent quantity of total membrane proteins from Ncstn−/− MEFs stably expressing FLAG-tagged wild-type, mutant (V439G) NCSTN, or EV control. (E) γ-secretase activity was measured by mixing a FLAG-tagged mouse Notch1 or 2 substrate (N1- or N2-N100) with microsomal proteins isolated from NcstnV439G/V439G mice and wild-type littermates in the presence or absence of the γ-secretase inhibitor DAPT in vitro. N1-N100 and N2-N100 are membrane-tethered Notch fragments resulting from ectodomain shedding of Notch1 and 2, respectively. Both N1- and N2-N100 and their γ-secretase-cleaved product, NICD, were visualized by immunoblotting using an anti-FLAG antibody. Data are representative of three independent experiments.