Sema3A stimulation induces redistribution of Fas into lipid rafts. (A) Jurkat cells (107) were treated with control IgG or Sema3A-Fc for 60 minutes and subjected to density gradient fractionation. Fractions were immunoblotted with antibodies for Lck, Fas, FADD, caspase-8, and NP1. Fractions corresponding to lipid raft (R) and soluble (S) proteins are indicated. (B) Jurkat cells were preincubated with the blocking anti-NP1 antibody or a control Ab. Then, cells were treated as in panel A. Fractions 4 (R) and 11 (S) were immunoblotted with antibodies specific for GM1 and Fas. Data in panels A and B are representative of at least 3 independent experiments. (C) Jurkat cells were stimulated with Sema3A-Fc for 60 minutes, depleted of cholesterol with 15 μg/mL MBCD for 10 minutes at 37°C, and then stimulated with CH11 for 6 hours. Cell death was determined by flow cytometry with annexin V staining. P value is shown.
