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Jurkat cells were transfected with an episomal vector and clones selected in hygromycin-containing medium. Clone J14 lacked TCR-mediated CD69 up-regulation, independently of the episomal vector, which was subsequently lost during growth in nonselective media. Absence of integration of the episomal DNA was confirmed by Southern blotting.
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D. Yablonski, M. R. Kuhner, T. Kadlecek, A. Weiss, data not shown.
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25
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Flag-tagged SLP-76 (Sal I-Xba I fragment) (19) was subcloned into pAWneo3′, which bears the neomycin-selectable marker. J14 cells were stably transfected with the resulting plasmid or the pAWneo3′ vector by standard procedures
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Flag-tagged SLP-76 (Sal I-Xba I fragment) (19) was subcloned into pAWneo3′, which bears the neomycin-selectable marker. J14 cells were stably transfected with the resulting plasmid or the pAWneo3′ vector by standard procedures.
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No significant differences were found between J14 and the vector-transfected subclones nor between different SLP-76-transfected subclones in any of the assays described; therefore, representative results are shown throughout
-
No significant differences were found between J14 and the vector-transfected subclones nor between different SLP-76-transfected subclones in any of the assays described; therefore, representative results are shown throughout.
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40
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2642701770
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The NFAT and IL-2-luciferase reporter constructs were provided by G. Crabtree (Stanford University)
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The NFAT and IL-2-luciferase reporter constructs were provided by G. Crabtree (Stanford University).
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41
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note
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Cells were mock stimulated with phosphate-buffered saline (no stimulation) or stimulated with C305 ascites (1:500), rapidly collected, and lysed in NP-40 lysis buffer as described (9). Whole-cell lysates or washed immune complexes were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Immobilon-P (Millipore) and probed with primary and secondary antibodies as described (9).
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2642677703
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Antibodies to ZAP-70, ltk, and LAT have been described (6, 24).
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Antibodies to ZAP-70, ltk, and LAT have been described (6, 24).
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46
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2642710096
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note
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We thank G. Koretzky for providing us with anti-serum to human SLP-76 and the SLP-76 cDNA and for communicating results before publication, L. Samelson for the antiserum to LAT, N. van Oers for the monoclonal antibodies to Vav, C. Liao for the antiserum to ltk, and the Weiss lab for their discussions and comments. Supported in part by a grant from the National Cancer Institute, CA72531.
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