American Society of Hematology

Figure 6.

$Figure 6. Arf6 mediates PI5KIα membrane recruitment and PLD activation upon CD16 stimulation. (A) PI5KIα immunoprecipitates were obtained from protein extract of membrane and soluble fraction of unstimulated or CD16-stimulated cultured human NK cells. Immunoprecipitated samples were divided into 2 equal aliquots and loaded on 12% (top) or 8% (bottom) SDS-PAGE and analyzed by immunoblotting as indicated. (B) Protein extracts derived from membrane and soluble fraction of unstimulated or CD16-stimulated cultured human NK cells were loaded on 12% SDS-PAGE and analyzed by immunoblotting. (C) Arf6-WT recombinant vaccinia virus–infected NK cells were stained with anti-HA mAb and analyzed by confocal microscopy (original magnification, × 600). (D) 3H oleic acid–radiolabeled NK cells were left uninfected or infected as indicated. Cells were left unstimulated (□) or stimulated with anti-CD16 (▪) in the presence of 0.5% ethanol. Lipids were separated by TLC, and the percentage of counts per minute of PetOH with respect to the total counts per minutes of phospholipids was quantified by liquid scintillation counted as a measure of PLD activity. Results are expressed as arbitrary units. Data from 3 independent experiments ± SD is shown.$

Arf6 mediates PI5KIα membrane recruitment and PLD activation upon CD16 stimulation. (A) PI5KIα immunoprecipitates were obtained from protein extract of membrane and soluble fraction of unstimulated or CD16-stimulated cultured human NK cells. Immunoprecipitated samples were divided into 2 equal aliquots and loaded on 12% (top) or 8% (bottom) SDS-PAGE and analyzed by immunoblotting as indicated. (B) Protein extracts derived from membrane and soluble fraction of unstimulated or CD16-stimulated cultured human NK cells were loaded on 12% SDS-PAGE and analyzed by immunoblotting. (C) Arf6-WT recombinant vaccinia virus–infected NK cells were stained with anti-HA mAb and analyzed by confocal microscopy (original magnification, × 600). (D) ³H oleic acid–radiolabeled NK cells were left uninfected or infected as indicated. Cells were left unstimulated (□) or stimulated with anti-CD16 (▪) in the presence of 0.5% ethanol. Lipids were separated by TLC, and the percentage of counts per minute of PetOH with respect to the total counts per minutes of phospholipids was quantified by liquid scintillation counted as a measure of PLD activity. Results are expressed as arbitrary units. Data from 3 independent experiments ± SD is shown.

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