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T cells from BALB/c lymph nodes were purified (15) and cultured for 60 hours in a dish coated with mAb to CD3 (anti-CD3) (145-2C11; 2 μg/ml) (16) and mAb to CD28 (anti-CD28) (PV-1; 2 μg/ml) (17). Cells were then washed three times to remove any residual antibodies present on the cell surface and rested at 37°C for additional 4 hours.
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Immunoprecipitations were performed with lysates prepared in LB [1% Nonidet P-40, 50 mM tris-HCI (pH 7.4), 150 mM NaCl, 20 mM EDTA (pH 8.0), 1 mM sodium vanadate, leupeptin (10 μg/ml), 10 μM aprotinin, 1 mM phenylsulfonylsulfoxide]. Lysates were precleared twice with protein A beads and once with protein A beads coated with a mAb from a control hamster (UC3-10A6) (I8) before precipitating antibodies were added. Immunoprecipitation was performed overnight at 4°C. Immune complexes were washed five times with LB and subjected to SDS-PAGE.
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+ by fluorescence-activated cell sorter analysis.
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The CTLA-4 tyrosine mutant (Y201F/Y218F) was generated by site-directed mutagenesis (Chameleon site-directed mutagenesis kit, Stratagene) and the presence of mutations was verified by DNA sequencing. A cDNA sequence incorporating the coding region of murine TCRζ was generated by reverse transcription and polymerase chain reaction from RNA extracted from activated murine T cells and cloned into the mammalian expression vector pCDNA3.1(+).
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note
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Supported by the National Institutes of Health (PHS PO1 AI35294-6). Additional support was provided by a Howard Hughes Medical Institute Medical Student Research Training Fellowship (D.K.H.) and a Mayo Foundation Scholarship (M.G.). We thank G. Feng for the reagents; A. Sharpe for CTLA-4 knockout mice; N. Patel and C. Long for technical assistance; S. Thomas, M. Fonstein, and W. Buikema for services provided at the Cancer Research Center (CRC) DNA Sequencing Facility; and M. Clark for critical reading of the manuscript.
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