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A porcine alveolar macrophage cDNA library in λACT2 (5) was cotransformed with A238L cloned in pCBT9 (Clontech) in yeast strain Y190. Clones encoding interacting proteins were selected on medium lacking histidine and by expression of β-galactosidase (β-Gal). The sequence of inserts was determined.
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0026568735
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A238L and I14L were tagged with the influenza virus HA epitope; A238L and IκB with the simian virus 5 (SV5) epitope [T. Hanke, P. Szawlowski, R. E. Randall, J. Gen. Virol. 73, 653 (1992)] in pCDNA3 (Invitrogen). In vitro-translated, radiolabeled proteins were mixed with 2 μg of CaN (Sigma), 2 μg of CypA (Sigma) and 10 μM CsA (Sandoz) where appropriate, absorbed with protein A-Sepharose (Sigma), and immunoprecipitated with 2 μg of anti-HA (Boehringer) or 5 μg of anti-SV5 (Serotec). Complexes were analyzed by autoradiography or protein immunoblot analysis with polyclonal anti-CaN (Chemicon) or monoclonal anti-CaN(B) (Sigma).
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Vero cells were infected with wild-type BA71V ASFV or SV5-A238L recombinant ASFV (12) for 4 to 10 hours. BSC1 cells were infected with modified vaccinia ankara (MVA) expressing T7 RNA polymerase [G. Sutter, M. Ohlmann, V. Erfle, FEBS Lett 371, 9 (1995)] and transfected with pT7-SV5-A238L or pT7-SV5-IκB. Proteins were radiolabeled, and the lysed cell extracts were immunoprecipitated and analyzed as before (9).
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Sutter, G.1
Ohlmann, M.2
Erfle, V.3
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Regions flanking the A238L ORF were cloned into KS vector (Stratagene), and the β-Gal gene downstream from the ASFV vp72 promoter [J. M. Rodriguez, F. Almazan, E. Vinuela, J. F. Rodriguez, Virology 188, 67 (1992)] was cloned between them. In addition, SV5-tagged A238L was cloned downstream from the A238L promoter. Recombinant ASF viruses ΔA238L and SV5-A238L were isolated by using X-Gal (5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside). Recombinant baculovirus expressing A238L (A238L-Bac) was constructed with use of the BAC To BAC baculovirus expression system (Life Technologies). Anti-A238L was raised in rabbits by using bacterially expressed A238L purified by SDS-polyacrylamide gel-electrophoresis.
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2642653294
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note
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25. PCR-amplified fragments corresponding to nucleotides 1309 to 1669 of the NFATc ORF (GenBank U08015) were cloned into pT7-Blue2 (Novagen), and the nucleotide sequence was determined.
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23
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2642590070
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note
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The A238L ORF was cloned in pCDNA3. The reporter plasmids used were NFAT-luc, mutant NFAT-luc (mNFAT-luc), and AP-1-luc. Transfected RS-2 cells were treated after 16 hours with (where appropriate) 40 nM PMA (Sigma), 4 μM ionomycin (Sigma), and 1 μM CsA (Sandoz), and luciferase activity was assayed 24 hours later.
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24
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Franz, B.1
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2642624710
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note
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We thank S. J. Elledge for two-hybrid reagents; R. Hay for PT7-SV5-IκB; G. R. Crabtree for the NFAT-luc reporter plasmid; E. McKenzie, I. O'Beirne, and A. Lennard for mNFAT-luc and AP-1-luc; and Sandoz Pharmaceutical for CsA. Supported by the Biotechnology and Biological Sciences Research Council and Ministry of Agriculture Fisheries and Food.
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