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16-CG in PBS on day 50 after the primary immunization. The serum concentrations of NP- and CG-specific antibodies were determined at different times after immunization as described (27).
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2 fragment goat anti-mouse IgM (2.5 μG/ml) (Dianova, Germany) as described (26). Data were evaluated by t test, with the use of Statistica software.
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51
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2 goat antimouse IgM (25 μg/ml) at 37°C and the reaction was stopped at different times by placement of cells on ice. Cells were lysed for 20 min at 4°C in lysis buffer containing 1% NP-40; 20 mM triethanolamine (pH 7.8); 150 mM NaCl; 500 mM EDTA; 100 mM orthovanadate; and a mixture of protease inhibitors-PMSF, pepstatin, leupeptin, and antipain at standard concentrations (5, 26). For the analysis of Btk phosphorylation. Btk was precipitated from cell lysates with a rabbit polyclonal antiserum raised against a peptide corresponding to amino acids 71 to 93 of Btk. The specificity of the immunoprecipitation was controlled by incubation of cell lysates with the antiserum to Btk in the presence of the immunogenic peptide for 1 hour. The phosphorylation of Btk was determined by protein immunoblot analysis of immunoprecipitates with the antibody to phosphotyrosine 4G10 and was detected with ECL reagents (Amersham). All samples contained equal amount of immunoreactive protein as determined by immunoblot analysis of immunoprecipitates with antibodies to Btk.
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57
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9444258449
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We thank A. Benner, C. Uthoff-Hachenberg, and C. Göttlinger for technical assistance; P. Parker for the antibodies to PKC-βll; R. Kühn for E14.1 ES cells; K. Rajewsky for support and helpful discussions; and R. Pelanda, R. Rickert, R. Torres, and N. Wagner for critical reading of the manuscript. Supported by the Bundesministerium für Forschung und Technologie (BMFT grant 0316150A) (S.St.), the Deutsche Forschungsgemeinschaft through Sonderforschungsbereich 243 (A.T.), and the Fonds der Chemischen Industrie (C.S.).
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