American Society of Hematology

Figure 4.

$Figure 4. ART2.2-GPI segregates with the lipid raft fraction in sucrose gradients. DC27.10 cells stably transfected with ART2.2-GPI (A) or ART2.2-Tm (B) were solubilized by ultrasonication in 1% TX-100 at 4°C. Lysates were subjected to sucrose gradient centrifugation. Isopycnic equilibrium was achieved by centrifugation at 200 000g for 14 hours. Twelve fractions were harvested from the top of the gradient. Fractions 1-3, 6-7, and 8-9 were pooled. Fraction 4 contained the visible opaque band at the 5%/30% sucrose interface. The pellet at the bottom of the tube was resuspended by ultrasonication in 2% SDS. Proteins in individual or pooled fractions were precipitated and were subjected to SDS-PAGE immunoblot analysis using anti-ART2.2 mAbs 9-13/10-6, anti-Gβ, pAb, T20, and CT. Total protein in each fraction was visualized by silver staining (C). Results are representative of 3 independent experiments.$

ART2.2-GPI segregates with the lipid raft fraction in sucrose gradients. DC27.10 cells stably transfected with ART2.2-GPI (A) or ART2.2-Tm (B) were solubilized by ultrasonication in 1% TX-100 at 4°C. Lysates were subjected to sucrose gradient centrifugation. Isopycnic equilibrium was achieved by centrifugation at 200 000g for 14 hours. Twelve fractions were harvested from the top of the gradient. Fractions 1-3, 6-7, and 8-9 were pooled. Fraction 4 contained the visible opaque band at the 5%/30% sucrose interface. The pellet at the bottom of the tube was resuspended by ultrasonication in 2% SDS. Proteins in individual or pooled fractions were precipitated and were subjected to SDS-PAGE immunoblot analysis using anti-ART2.2 mAbs 9-13/10-6, anti-Gβ, pAb, T20, and CT. Total protein in each fraction was visualized by silver staining (C). Results are representative of 3 independent experiments.

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