American Society of Hematology

Fig. 5.

$Fig. 5. Immunoprecipitation of PI 3-kinase activity with 14-3-3 or GP Ib from platelet cytosol. / (A) Six hundred microliters of human platelet cytosol (5 mg/mL) was immunoprecipitated using 5 μL nonimmune, anti-p85, anti–14-3-3ζ, or antiglycocalicin antiserum, captured on protein A–Sepharose, and washed, and PI 3-kinase activity was determined using PtdIns as a substrate as described in “Materials and methods.” (B) The PtdIns 3-P formed in immunoprecipitates of nonstimulated platelet Triton-soluble cytosolic fractions using the indicated antibodies was expressed as a percentage of that observed in p85 immunoprecipitates (IP). Values represent the mean ± SD of 4 experiments. (C) Platelets were stimulated for 5 minutes with thrombin (1 U/mL), and PI 3-kinase assays were performed on washed immunoprecipitates using the indicated antibodies. The decrease in enzyme activity is expressed as a percentage relative to that observed in nonstimulated immunoprecipitates using the same antibody. Values represent mean ± SD of 4 experiments.$

Immunoprecipitation of PI 3-kinase activity with 14-3-3 or GP Ib from platelet cytosol.

(A) Six hundred microliters of human platelet cytosol (5 mg/mL) was immunoprecipitated using 5 μL nonimmune, anti-p85, anti–14-3-3ζ, or antiglycocalicin antiserum, captured on protein A–Sepharose, and washed, and PI 3-kinase activity was determined using PtdIns as a substrate as described in “Materials and methods.” (B) The PtdIns 3-P formed in immunoprecipitates of nonstimulated platelet Triton-soluble cytosolic fractions using the indicated antibodies was expressed as a percentage of that observed in p85 immunoprecipitates (IP). Values represent the mean ± SD of 4 experiments. (C) Platelets were stimulated for 5 minutes with thrombin (1 U/mL), and PI 3-kinase assays were performed on washed immunoprecipitates using the indicated antibodies. The decrease in enzyme activity is expressed as a percentage relative to that observed in nonstimulated immunoprecipitates using the same antibody. Values represent mean ± SD of 4 experiments.

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