American Society of Hematology

Figure 5.

$Figure 5. Role of proline-rich domain in FasL lipid raft localization. (A) Schematic organization of the FasL cytoplasmic tail. Positions of FasL cytoplasmic mutants are indicated. Putative signaling domains are in bold. TM indicates transmembrane domain. (B) 293T cells were transfected with full-length or different FasL cytoplasmic deletion mutants (Δ37, Δ67) and lipid rafts were isolated using the short method. FasL, flotillin, and tubulin were detected by Western blot. NR indicates nonraft fraction; R, raft fraction. The percentage of FasL in lipid rafts was determined densitometrically. A typical experiment of 5 is shown. (C) 293T cells were transfected with full-length FasL or deletion mutants and cell surface FasL expression was detected by flow cytometry. Shaded histograms, isotype control; black lines, anti-FasL staining. Numbers indicate mean fluorescence intensities of triplicates ± SD. (D) FasL activity was measured by induction of target cell killing. Target cell killing was normalized for unequal cell surface expression of full-length and mutant FasL constructs (C) by dividing percent DNA fragmentation with the mean fluorescence intensity of FasL cell surface expression. A typical experiment of 4 is shown.$

Role of proline-rich domain in FasL lipid raft localization. (A) Schematic organization of the FasL cytoplasmic tail. Positions of FasL cytoplasmic mutants are indicated. Putative signaling domains are in bold. TM indicates transmembrane domain. (B) 293T cells were transfected with full-length or different FasL cytoplasmic deletion mutants (Δ37, Δ67) and lipid rafts were isolated using the short method. FasL, flotillin, and tubulin were detected by Western blot. NR indicates nonraft fraction; R, raft fraction. The percentage of FasL in lipid rafts was determined densitometrically. A typical experiment of 5 is shown. (C) 293T cells were transfected with full-length FasL or deletion mutants and cell surface FasL expression was detected by flow cytometry. Shaded histograms, isotype control; black lines, anti-FasL staining. Numbers indicate mean fluorescence intensities of triplicates ± SD. (D) FasL activity was measured by induction of target cell killing. Target cell killing was normalized for unequal cell surface expression of full-length and mutant FasL constructs (C) by dividing percent DNA fragmentation with the mean fluorescence intensity of FasL cell surface expression. A typical experiment of 4 is shown.

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