Alternatively, once the substrate, a counterpart of AICD possibly, is bound to-secretase, it is cleaved at the A40 preferentially, A43, or A46 site, all aligning at one particular -helical surface of CTF, or at the A45 or A42 site, both aligning at the opposite surface (Fig. wtAPP695 and mouse neuroblastoma N2a cells expressing Swedish mtAPP695, provided by Drs kindly. C. Haass (Ludwig-Maximilians University, Munich, Germany) and S. S. Sisodia (University of Chicago, Chicago, IL), respectively, were cultured as described previously (Citron et al., 1992; Thinakaran et al., 1996). The 7WD10 cells were transfected stably with wt or various mtPS1/2 cDNAs (Qi et al., 2003). The pcDNA4/TO vector (Invitrogen, Carlsbad, CA) with SP-DA-CTF1-99 cDNA insert (Lichtenthaler et al., 1999a) was transfected into T-Rex-CHO cells (Invitrogen) using Lipofectamine2000 (Invitrogen), and the stable cell lines were selected using 500 g/ml Zeocin (Invitrogen). Expression of CTF was induced by the addition of 1 g/ml tetracycline (Invitrogen) to the culture media. Dominant-negative (DN) mtPS1 (D257A/D385A) (Wolfe et al., 1999) cDNA was generated using the Quick Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). The pIRESneo3 vectors (BD Biosciences, Palo Alto, CA) with wt and DN mtPS1 cDNA inserts were transfected into T-Rex-CHO cells stably expressing CTF, and GATA3 stable cell lines were selected with 500 g/ml G418 sulfate. The plasmids of wtAPP695, APP695 bearing a di-lysine endoplasmic reticulum (ER) retention signal (Jackson et al., 1990), and APP695 carrying the CHO cells expressing CTF were incubated with a -secretase inhibitor inducibly, either {1Synthetic A peptides (DAEFRHDSGYEVHHQK and DAEFR) were conjugated to thyroglobulin through Cys at the C termini. The former was used for primary immunization with an adjuvant, whereas the latter was Trilostane used for booster injections. Hybridomas were produced by polyethylene glycol-mediated fusion between immunized splenocytes and X63-Ag8-653 (Kinebuchi et al., 1991), and the clone 82E1 was selected using peptide-coated immunoplates. Other monoclonal antibodies against A that were used were 6E10 (raised against A1-17), 4G8 (epitope: A17-24; Signet Laboratories, Dedham, MA), and BAN50 (raised against A1-16) (Suzuki et al., 1994). The polyclonal antibodies against the cytoplasmic domain of APP were UT421 (Tomita et al., 1998) and C4 (Takio et al., 1989). The polyclonal antibodies against PS1 (anti-G1L3) were described previously (Tomita et al., 1999). The conditioned media for 6-8 hr culture were incubated with BAN50 at 4C for 6 hr. Harvested cells were lysed with Tris-buffered saline (TBS) (in mm: 50 Tris-HCl, pH 7.6, 150 NaCI, 1 EGTA, Trilostane and 1 EDTA) containing 1% Triton X-100 and various protease inhibitors (0.1 mm diisopropyl fluorophosphate, 0.1 mm phenylmethylsulfonyl fluoride, 5 g/ml N-ptosyl-l-lysine chloromethyl ketone, 1 g/ml antipain, 1 g/ml pepstatin, 1 g/ml leupeptin, 1 g/ml bestain, 1 g/ml amerstain, 5 mm 1,10-phenanthroline monohydrate, and 10 mm thiorphan). The homogenates were cleared by centrifugation at 540,000 for 20 min. The cell lysates were first immunoprecipitated with C4-bound protein G-Sepharose at 4C for 1 hr to remove full-length APP and CTF, and the resultant supernatants were incubated with BAN50 at 4C for 6 hr further. The immune complexes were collected with protein G-Sepharose and eluted with the Laemmli SDS sample buffer. The immunoprecipitated proteins were separated on Tris/Tricine/8 m urea gels, followed by Western blotting with 82E1. For each set of the experiments, each supernatant was diluted to the same protein concentrations appropriately, and an equal volume of the supernatant was used for immunoprecipitation. The cerebra of 2.5-month-old male Tg2576 mice (IBL, Fujioka, Japan) (Hsiao et al., 1996) were homogenized in 5 vol of TBS buffer containing protease inhibitors. The homogenate was centrifuged at 540,000 for 20 min to obtain a TBS-soluble fraction. After washing with the same buffer, the resultant pellet was homogenized in 5 vol of TBS buffer containing 1% Triton X-100 and protease inhibitors, and the homogenate was centrifuged at 540,000 for 20 min to obtain a Triton-soluble fraction. The resulting pellet was suspended in 1 vol of guanidine hydrochloride (GuHCI) by sonication. The suspension was centrifuged at 265,000 for 20 min to obtain a GuHCI-soluble fraction, which Trilostane was diluted 12-fold. Each fraction was subjected to immunoprecipitation with BAN50, as described.
Alternatively, once the substrate, a counterpart of AICD possibly, is bound to-secretase, it is cleaved at the A40 preferentially, A43, or A46 site, all aligning at one particular -helical surface of CTF, or at the A45 or A42 site, both aligning at the opposite surface (Fig
