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Fig. 5. Temperature-dependence of tyrosine phosphorylation and ERK activation in Jurkat cells. Cells were incubated at the indicated temperatures for 10 minutes, of which, anti-CD3 (OKT3) at 1 µg/ml was present for the last 5 minutes in the stimulated aliquots (lanes +). Lysates were analysed by western blotting for (A) tyrosine phosphorylation (4G10) or (B) ERK activation (anti-phospho-p44/42 MAP kinase). Gels were loaded on an equal protein basis. (C) Cells incubated at 10°C for 5 minutes were stimulated with 1 µg/ml OKT3 for 5 minutes at 10°C and lysed in 1% NP40-containing buffer. Precleared lysates were used for immunoprecipitation with either anti-ZAP-70, anti-LAT or anti-Lck rabbit antisera coupled to protein A Sepharose beads with dimethylpimelidate. Precipitated proteins were recovered by glycine elution and analysed by western blotting for tyrosine phosphorylation. The arrow indicates the band in the Lck lane. Molecular mass markers are indicated (kDa). Fluorographs shown are representative of 11, nine and three experiments, respectively.
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