Ligand-independent partitioning of FasL in lipid membrane rafts. (A) PNSs from HEK293 cells stably expressing human FasL were prepared and then solubilized in Brij 98 before they were subjected to sucrose gradient separation, as described in “Materials and methods.” Sucrose fractions were analyzed by Western blot with anti-FasL, anti-Fyn, and anti-Rab5 antibodies. Detergent-insoluble membrane fractions containing isolated rafts were identified by the presence of the raft marker protein Fyn. Nonraft components of the membrane (eg, Rab5) were purified in the heavy fraction (HF). (B) An experiment similar to that described in panel A was performed with Jurkat cells (J16) stably expressing hFasL. Again, a significant fraction of FasL was detected in raft fractions. (C-D) Brij 98 solubilization and sucrose gradient separation revealed that in the natural killer cell line NK92 (C) and in activated primary human T cells (D), endogenous FasL was localized within detergent-insoluble rafts. Immunoblots were performed on pooled heavy (8-9; HF) and light (1-4; Raft) fractions.