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-l- phenotype.
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21
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0345394507
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MAP kinase activation in T lymphocytes also occurs through GRB-2-mediated pathways (21). However, tyrosine-phosphorylated proteins bound by GRB-2's SH2 domain, including Cbl, Slp-76, and LAT (21, 22), were normal in mutant cells (8), suggesting intact GRB-2-mediated signals.
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0344532219
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6) were stimulated for indicated times with plate-bound 2C11 (7.5 μg/ml), lysed in 1% Triton and 0.1% SDS in phosphate-buffered saline with protease inhibitors (Boehringer Mannheim) and sodium vanadate, and immunoprecipitated with either anti-TCR zeta chain (gift of L. Samelson) or anti-Zap-70 (Upstate). Immune complexes were resolved by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-phosphotyrosine (4G10 Upstate).
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0344963100
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pMC1neo and multiple stop codons were inserted into the Kpn I site of rlk (encoding the start of the SH2 domain) with 6-kb upstream and 3-kb downstream sequences, followed by herpes simplex virus thymidine kinase (HSV-TK). The construct was electroporated into TC1 ES cells and G418-gancyclovir-resistant colonies screened by Southern (DNA) blot analyses with Hind III and a flanking 3′ probe. Gene disruption at the 5′ end was confirmed with an internal probe.
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Splenocytes were stimulated with PMA (10 ng/ml) and ionomycin (1 μg/ml) for 24 hours to ensure equal proliferation, washed, and grown with IL-2 for 3 days. Cells were restimulated with anti-CD3ε (2C11) for 24 hours, and cell viability was determined by flow cytometry.
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6 per milliliter) loaded with Fluo-3-AM (5 μg/ml) and Fura-Red-AM (5 μg/ml) (Molecular Probes, Eugene, OR) (13) was analyzed with MultiTime Kinetic Experiment Analysis Software (Phoenix Flow Systems, San Diego, CA), and data were expressed as percentage responding relative to unstimulated cells.
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3 Radioreceptor Assay Kit (NEN Life Science).
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0344963098
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We thank L Samelson, J. O'Shea, J. Dodson, and R. Nussbaum for comments; A. Wynshaw-Boris, C. Canna, and D. Bernards for assistance with ES cells; S. Anderson for flow cytometry; and M. Czar, A. Venegas, W. Hively, G. Elliot, M. Chamorro, L. Brody, E. Arnold, M. Erdos, L. Zheng, B. Combediere, and R. Siegel. Animal care was in accordance with NIH guidelines. This work was initiated in the laboratory of M.J.L., continued in the laboratory of H.E.V., and completed in the laboratory of P.L.S. E.M.S. and J.D. are Howard Hughes Medical Institute-NIH Research Scholars.
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