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note
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The mouse BAD cDNA containing the coding sequence was amplified by reverse transcription polymerase chain reaction (PCR) with specific primers (5′-AAAGATCTAGAATGGGAACCCCAAAGCAGCCCTCGCTG-3′ and 5′-TTGAATTCACTGGGAGGGGGTGGAGCCTCCTTTG-3′) and ligated in frame into the pcDNA3 vector (Invitrogen) containing an AU1-tag epitope. The authenticity of all constructs was confirmed by dideoxy sequencing. FL5.12 cells were transfected by electroporation (960 μF, 250 V) with the pcDNA3-AU1-BAD construct alone or in combination with a human Bcl-2-expressing plasmid (pSFFV-Flag-Bcl-2) and selected with G418 (1 mg/ml). After selection, expression was assessed in the bulk population as well as in independent clones by flow cytometry.
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85069031499
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note
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136 to Ala mutations was generated by site-directed mutagenesis with the use of PCR (5′- CAGTGCGTACCCAGCGGGGACCGAGGAGGATGAAGGGTAGGAGGAGGAGTCTAGCCCTTTTCGAGGACGCTCGCGTGCGG CTCCC-3′ and 5′- GGGAGCCGCACGCGAGCGTCCTCGAAAAGGGCTAAGCTCCTCCTCCATCCCTTCATCCTCCTCGGTCCCCGCTGGGTAGC GACTG-3′), then subcloned into the pET-30a(+) plasmid and purified as WT rBAD. The authenticity of all constructs was confirmed by dideoxy sequencing.
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85069023645
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note
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2, 1 mM EGTA, aprotinin (2 μg/ml), leupeptin (2 μg/ml), 1 mM phenylmethylsulfonyl fluoride, and 50 mM NaF]. BAD was immunoprecipitated with an antibody to AU1 (Babco); immunocomplexes were recovered with protein G-Sepharose and resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred to a nitrocellulose membrane and exposed to a Phosphorlmager screen (Molecular Dynamics) to quantitate the radioactivity incorporated into the BAD protein.
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85069009478
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Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; F, Phe; G, Gly; H, His; I, Ile; M, Met; N, Asn; P, Pro; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr
-
Single-letter abbreviations for the amino acid residues are as follows: A, Ala; E, Glu; F, Phe; G, Gly; H, His; I, Ile; M, Met; N, Asn; P, Pro; R, Arg; S, Ser; T, Thr; V, Val; and Y, Tyr.
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note
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We thank T. F. Franke, M. Andjelkovich, and P. Tsichlis for reagents and R. Perez-Ballestero, M. Benedict, and N. Inohara for valuable discussions and critical review of the manuscript. Supported in part by NIH grant CA-64556, the University of Michigan/Parke-Davis Fellowship Program (L.d.P.), a Spanish Ministry of Science and Education Postdoctoral Fellowship (M.G.-G.), and an NIH Research Career Development Award (G.N.)
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