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For cell-free translation of PrP and mutants, each coding region was engineered into an sp64 plasmid vector containing the SP6 promoter followed by the 5' untranslated region of Xenopus globin. Transcription with SP6 polymerase, translation in rabbit reticulocyte lysate containing microsomal membranes from dog pancreas, and proteolysis were done as described previously [(34), and references therein]. All translation reactions were carried out at 32°C for 60 min and proteolysis reactions at 0°C for 60 min with PK (0.5 mg/ml). Products were immunoprecipitated with either the 3F4 mAb (Fig. 1, D and E) or R073 antibody (Fig. 1F) (35) and analyzed by 10% tricine-SDS-PAGE (36), and the proteins were visualized by autoradiography.
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NtmPrP.
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54
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6844224721
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note
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For transgenic mice production, PrP coding regions were engineered into the cosSHa.Tet cosmid expression vector (37) at the Sal I site. cosSHa.Tet cosmids containing the appropriate PrP transgenes were digested with Not I, and the transgene was purified and used for microinjection into the pronuclei of fertilized FVB/PrnpO/O oocytes as previously described (38). We assessed the presence of the transgene in weanling animals by screening genomic DNA isolated from tail tissue with a probe specific to the 3′ untranslated region of the SHaPrP gene contained in the cosSHa.Tet vector. PrP expression was assessed by immunoblotting of brain tissue homogenate with 13A5 mAb, comparing this tissue with serial dilutions of normal Syrian hamster brain tissue (see Rg. 2C for an example). Levels of expression (relative to normal hamster) of each transgenic line are shown in Table 1.
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55
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note
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2, 50 mM Hepes (pH 7.5), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] by successive passage, five times each, through 16-, 18-, 20-, and 22-gauge needles. Homogenate was centrifuged for 10 min at 12,000g at 4°C. Supernatant was removed and centrifuged for either 20 min at 100,000 rpm in TLA-100.3 rotor (Beckman) or 60 min at 65,000 rpm in 70.1Ti rotor (Beckman). Supernatant was discarded and pellet resuspended in the same buffer as above, but without PMSF, at a concentration of 500 μl per gram of starting tissue. Topology was assessed by digestion of membranes with PK as described (39), followed by digestion with PNGase as directed by the manufacturer (New England Biolabs). Samples were precipitated with trichloroacetic acid, separated by 10% tricine-SDS-PAGE, transferred to nitrocellulose, and probed with either 3F4 or 13A5 mAb as indicated in the figure legends.
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data not shown
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CtmPrP will be reported elsewhere (28). The proteolysis reactions were terminated by adding PMSF to 5 mM, incubating an additional 5 min, and transferring the sample to 5 volumes of boiling 1% SDS and 0.1 M tris (pH 8.9). Brain-derived samples were then digested with PNGase as directed by the manufacturer, resolved by 10% tricine-SDS-PAGE, transferred to nitrocellulose, and probed with either the 3F4 (Fig. 6B) or 13A5 (Fig. 6A) mAb. In vitro translation products (Fig. 5A) were immunoprecipitated with 3F4 and analyzed by SDS-PAGE and autoradiography.
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59
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note
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We thank J. Cayetano for help with the histopathology; A. Calayag, O. Nguyen, and R. Köhler for technical assistance; R. Cotter and C. Petromilli for animal care; H. Serban for reagents; G. Telling for advice; and J. Taltzelt, R. Nixon, S. Russel, and J. Lingappa for stimulating discussions. Supported by NIH grant AG02132 and the Medical Scientist Training Program.
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