Supplemental Data Nicastrin Functions as a γ-Secretase-Substrate Receptor

cDNAs and Antibodies Human cDNAs were used in this study unless indicated otherwise. Wild-type and mutant Nct-His, PS1, APH-1aL-His, HA-PEN-2, Flag-PEN-2, C99-Flag, nFlag-C99, N100-Flag/His, and His-Syt1 cDNAs were cloned into pFASTBac and baculoviruses were generated using the Bac-to-Bac Baculovirus Expression System (Invitrogen). pFASTBac/C99-Flag encodes C-terminally Flag-tagged C99 with a Met followed immediately by the β-secretase cleaved APP C-terminal fragment. pFASTBac/nFlag-C99 encodes a C99-derived polypeptide in which the seven residues of C99 after Met were replaced by the Flag epitope (DYKDDDDK). pFASTBac/N100-Flag/His encodes Val1711-Glu1809 of mouse Notch-1 and a C-terminal Flag/His tag. The bacterial expression construct pET21b/C99-Flag was a gift from Drs. M.J. LaVoie, D.J. Selkoe, and Y.M. Li (Esler et al., 2002; Li et al., 2000). To generate the construct for Cys-C99 (pHV-Cys), the His-tag and tobacco etch virus (TEV) recognition site sequence from pHisparallel was amplified and mutated via PCR to generate an NdeI/BamHI product with an internal NsiI site, changing the final Gly of the TEV recognition site to Cys. This fragment was then subcloned using the same sites into pET21b followed by insertion of C99-Flag as an NsiI/XhoI fragment. pCMV-IgG fusion constructs for mammalian cells were generated to contain the following sequences of various proteins fused to the Fc fragment of human IgG: nicastrin residues 1-50, nicastrin residues 1-669, fulllength α2δ1, and APP residues 1-205. pSGHV0-Nct(ECD) encodes nicastrin residues 34-669 in frame with the human growth hormone (hGH), an octahistidine tag (H8), and a cleavage site for TEV protease (Dann et al., 2001; Leahy et al., 2000). Wild-type and mutant nicastrin cDNAs in pcDNA6-V5/His and a cDNA for the APP signal peptide-fused C99 (SpC99) in pcDNA3 were generated as previously described (Lee et al., 2004; Luo et al., 2003; Yu et al., 2000). Antibodies used in this study include anti-sera for APH-1aL, PEN-2, PS1, and Nct (Lee et al., 2002; Lee et al., 2004; Luo et al., 2003), anti-cleaved Notch-1 (Val1744) antibody (Cell Signaling), anti-APP cytoplasmic domain (anti-APP-CTD), anti-biotin, antihuman IgG, anti-Flag (Sigma), anti-His (Qiagen), anti-HA (Santa Cruz), and anti-fluorescein/Oregon Green (Molecular Probes) antibodies.