American Society of Hematology

Fig. 4.

$Fig. 4. Association of PI3K with Lyn kinase in lipid bodies of PMN leukocytes. / (A) Localization of PI3K and Lyn kinase in lipid bodies of PAF-primed PMN leukocytes. PMN leukocytes were preincubated with PAF (0.5 μmol/L) for 1 hour at 37°C to induce lipid body formation before subcellular fractionation. Proteins were then concentrated from each subcellular fractions by TCA precipitation, and 20 μg from each fraction were electrophoresed and immunoblotted with an anti-PI3K p85 mAb and anti-Lyn kinase pAb. (B) Physical association of Lyn kinase with PI3K in lipid bodies of activated PMN leukocytes. PMN leukocytes were pretreated with arachidonic acid (20 μmol/L) for 30 minutes before subcellular fractionation. Lyn kinase in the lipid body fraction (1.0 mL) was sequentially immunoprecipitated with nonimmune control rabbit antibody twice (preclear 1 and 2), followed by immunoprecipitation with 5 μg of anti-Lyn kinase rabbit pAb. The precipitates were electrophoresed and immunoblotted with anti-PI3K p85 mAb and developed with HRP-conjugated goat antimouse antibody.$

Association of PI3K with Lyn kinase in lipid bodies of PMN leukocytes.

(A) Localization of PI3K and Lyn kinase in lipid bodies of PAF-primed PMN leukocytes. PMN leukocytes were preincubated with PAF (0.5 μmol/L) for 1 hour at 37°C to induce lipid body formation before subcellular fractionation. Proteins were then concentrated from each subcellular fractions by TCA precipitation, and 20 μg from each fraction were electrophoresed and immunoblotted with an anti-PI3K p85 mAb and anti-Lyn kinase pAb. (B) Physical association of Lyn kinase with PI3K in lipid bodies of activated PMN leukocytes. PMN leukocytes were pretreated with arachidonic acid (20 μmol/L) for 30 minutes before subcellular fractionation. Lyn kinase in the lipid body fraction (1.0 mL) was sequentially immunoprecipitated with nonimmune control rabbit antibody twice (preclear 1 and 2), followed by immunoprecipitation with 5 μg of anti-Lyn kinase rabbit pAb. The precipitates were electrophoresed and immunoblotted with anti-PI3K p85 mAb and developed with HRP-conjugated goat antimouse antibody.

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