American Society of Hematology

Figure 4.

$Figure 4. Neutrophil apoptosis involves clustering of CD95 in lipid rafts. (A) CD95 is partially localized in lipid rafts in freshly isolated neutrophils. Neutrophils were lysed in Triton X-100–containing buffer at 4° C, and the lysate was fractionated on a sucrose density gradient to separate raft and nonraft proteins. Gradient fractions were analyzed by Western blotting for the presence of CD95. (B) Lipid rafts containing CD95 cluster in aged neutrophils. Neutrophils were cultured for 0 hours or 9 hours and then stained with FITC-conjugated cholera toxin B subunit to locate GM1 in lipid rafts and a nonactivating anti-CD95 antibody (ZB4) and a Texas red–conjugated anti–mouse IgG1 antibody. The images shown are representative of 3 separate experiments performed. (C) Neutrophil spontaneous apoptosis depends upon intact lipid rafts; disruption of lipid rafts inhibited neutrophil apoptosis. Neutrophils were cultured for up to 20 hours in the absence or presence of 10 μg/mL nystatin (○), 10 μg/mL filipin (•), or 0.1% methanol as solvent control (□), and apoptosis determined by analysis of cell morphology. (D) Assembly of the DISC complex depends on intact lipid rafts. Neutrophils were cultured with and without nystatin, lysed, and incubated with an anti-FADD antibody. FADD immunoprecipitates were then analyzed by Western blotting for the presence of CD95 and pro-caspase 8. The blots shown are representative of 3 separate experiments performed.$

Neutrophil apoptosis involves clustering of CD95 in lipid rafts. (A) CD95 is partially localized in lipid rafts in freshly isolated neutrophils. Neutrophils were lysed in Triton X-100–containing buffer at 4° C, and the lysate was fractionated on a sucrose density gradient to separate raft and nonraft proteins. Gradient fractions were analyzed by Western blotting for the presence of CD95. (B) Lipid rafts containing CD95 cluster in aged neutrophils. Neutrophils were cultured for 0 hours or 9 hours and then stained with FITC-conjugated cholera toxin B subunit to locate GM1 in lipid rafts and a nonactivating anti-CD95 antibody (ZB4) and a Texas red–conjugated anti–mouse IgG1 antibody. The images shown are representative of 3 separate experiments performed. (C) Neutrophil spontaneous apoptosis depends upon intact lipid rafts; disruption of lipid rafts inhibited neutrophil apoptosis. Neutrophils were cultured for up to 20 hours in the absence or presence of 10 μg/mL nystatin (○), 10 μg/mL filipin (•), or 0.1% methanol as solvent control (□), and apoptosis determined by analysis of cell morphology. (D) Assembly of the DISC complex depends on intact lipid rafts. Neutrophils were cultured with and without nystatin, lysed, and incubated with an anti-FADD antibody. FADD immunoprecipitates were then analyzed by Western blotting for the presence of CD95 and pro-caspase 8. The blots shown are representative of 3 separate experiments performed.

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