Subcellular localization of PI3K to cytoplasmic lipid bodies of U937 cells.

Lipid body and other cellular fractions were isolated from U937 cells as described in “Materials and Methods.” Lipid body fractions were identified microscopically by their content of Nile red staining lipid bodies. Fractions were assayed for LDH and sulfatase C activities as cytosolic and microsomal markers, respectively (A). (B) Proteins in cellular fractions were quantified by micro BCA assay. For lipid labeling of lipid bodies, cells were preincubated with [¹⁴C]-AA before subcellular fractionation, and results represent the total [¹⁴C]-labeled lipid present in each fraction. Data are representative of 3 independent experiments. (C) Western blotting of specific proteins present in subcellular fractions. Proteins (20 μg) concentrated from each subcellular fraction by TCA precipitation were electrophoresed on a 10% SDS-PAGE gel and immunoblotted with anti-PI3K p85 mAb, anti-PI3K p110β pAb, anti-annexin VI mAb, anti-MAP kinase ERK3 pAb, and an anti-MAP kinase anti-pan ERK mAb. (D) PI3K specific activities in freshly isolated subcellular fractions from GM-CSF stimulated U937 cells. PI3K activity with PI as substrate was measured by the formation of PI³P as described in “Materials and Methods.”