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The anti-PS2Loop is a PS2-specific antibody raised against the large hydrophilic loop domain following predicted transmembrane domain six of PS2 (7). Protein Quantitation, SDS-polyacrylamide gel electrophoresis (PAGE; 4 to 20% or 16%), and protein immunoblotting were done as described (9) except that the lysis buffer [10mM tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.5% NP-40, 5 mM EDTA plus either 0.3% SDS or 1% Sarkosyl] contained protease inhibitors, allowing for the efficient extraction of the 20-kD COOH-terminal fragment from the detergent-resistant fractions. Except in Figs. 1B and 3C (4 to 20% gradient gel), all SDS-PAGE was performed with 16% gels.
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9844256164
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note
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The 26- and 20-kD CTFs were absent in the uninduced samples (Fig. 1, lanes 9 to 12) and could be immunoprecipitated with antibodies to the FLAG epitope (14), indicating that these fragments were derived from the PS2 transgene. The 26-kD fragment is most likely the normal 25-kD cleavage product plus the eight-amino acid FLAG epitope tag. In the previous study (9), the regulated 26-kD normal cleavage product was not detectable on protein immunoblots with the relatively insensitive antibody to FLAG. As shown for PS1 (7), the generation of the normal PS2-CTF appears to be a regulated endoproteolytic event. This is evidenced by the observation that levels of the 26-kD transgene-derived PS2-CTF were not considerably greater than those of the native 25-kD CTF in the uninduced cells. Additionally, levels of the native 25-kD CTF were diminished in the induced cells, suggesting that the production of the transgene-derived CTF led to an attenuation in the amount of endogenous CTF. A similar type of "replacement" phenomenon has been reported for PS1 in transgenic mice expressing human PS1 (7). In contrast, the alternative 20-kD PS2-CTF did not appear to be generated in a regulated fashion (9).
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Relative amounts of 20-kD PS2 fragments from the cells cotreated with 1 μM STS (18 hours) plus either zVAD or zDEVD (1 to 200 μM) were measured by transmittance analysis of the protein immunoblot as described (13, 35). Twenty-five micromolar zVAD was as effective as 100 μM zDEVD in inhibiting apoptosis-induced cleavage of PS2. The amounts of 20-kD fragments from the cells treated with STS atone were standardized as 100%.
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In addition to H4 human neuroglioma cells, all findings were confirmed in native SK-N-SC neuroblastoma cells (14).
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Alternative endoproteolysis sites for PS2 are localized within the domain after predicted transmembrane domain 5, encoded by exon 11 (formerly exon 10) (36).
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30
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9844253969
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note
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The point mutations in the potential aspartate cleavage sites of PS2 (D326A and D329A) and control aspartate (P308A) were introduced into a PS2 open reading frame by site-directed mutagenesis with Muta-Gene phagemid kit (Bio-Rad). The inducibte construct encoding the ful-length PS1 with 3′ FLAG epitope sequence was generated as deserted for PS2 (9). To establish the inducible PS1 cefe, we sequenced the resulting constructs and stably transfected them into the H4 human neuroglioma founder cell line as described for the PS2-inducible cells (9).
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9844244090
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note
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Values (mean ± SD, n = 30; *P < 0.05) representing the ratios of alternative:normal CTF in multiple wild-type and FAD mutant (N141I) PS2 clonal lines induced for 24 hours were determined (73, 36). Data from three independent experiments with five different wild-type and mutant PS2 clonal lines paired according to expression level were used.
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We thank G. Thinakaran and S. Sisodia for antibodies and J. Henderson for technical assistance. Supported by grants from the National Institute on Aging, National Institute of Neurological Disorders and Stroke, and the Metropolitan Life Foundation. R.E.T. is a Pew Scholar, D.M.K. is a French Foundation Fellow, and T.-W. K. is a recipient of a National Research Service Award.
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