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Volume 7, Issue 6, Pages 831-836 (December 1997) TRAIL Receptors 1 (DR4) and 2 (DR5) Signal FADD-Dependent Apoptosis and Activate NF-κB Pascal Schneider, Margot Thome, Kim Burns, Jean-Luc Bodmer, Kay Hofmann, Takao Kataoka, Nils Holler, Jürg Tschopp Immunity Volume 7, Issue 6, Pages 831-836 (December 1997) DOI: 10.1016/S1074-7613(00)80401-X

Figure 1 Association of TRAIL-R1 with TRAIL-R2 (Top) Immunoprecipitation of cell extracts from 293T cells, transfected with expression vectors for the cytoplasmic segments of TRAIL-R1 and TRAIL-R2, was performed using an anti-Flag antibody (precipitating Flag-TRAIL-R1 and Flag-TRAIL-R2, respectively). Samples were analyzed for coprecipitating Myc-TRAIL-R2 or Myc-Fas by Western blotting using anti-Myc antibodies. IP, immunoprecipitating. (Middle and bottom) The expression levels of Flag-TRAIL-R1, Flag-TRAIL-R2, Myc-TRAIL-R2, and Myc-Fas in the corresponding cell extracts. Immunity 1997 7, 831-836DOI: (10.1016/S1074-7613(00)80401-X)

Figure 2 TRAIL-R1 and TRAIL-R2 (but Not TRAIL-R3) Induce Apoptosis That Can Be Inhibited by Proteins Competing with FADD Recruitment (A) DNA fragmentation of 293T cells expressing TRAIL-R1, TRAIL-R2, or TRAIL-R3, respectively, was determined by analyzing cytoplasmic histone-containing DNA complexes. Full-length TRAIL receptors were transfected alone or in combination with expression vectors for equine herpes virus E8-FLIP, human FLIPL, or FADD-DN. Where indicated, 50 μM z-VAD-fmk was added to the cells 8 hr after the onset of transfection. As a positive control, TRAMP expression vector (2 μg) was transfected. Data are the means ± SEM of triplicate determinations. OD, optical density. (B) Apoptosis of 293T cells stably expressing TRAIL-R1. Parental (wt) and TRAIL-R1–expressing cells were mock transfected or transfected with an expression vector for FADD-DN for 10 hr and then treated with recombinant soluble TRAIL (sTRAIL) (solid bars, 500 ng/ml) for 14 hr. Cells were analyzed by phase contrast microscopy (right, sTRAIL-treated TRAIL-R1–expressing cells), and the percentage of apoptotic cells was determined (left). Controls shown are cells not exposed to sTRAIL (open bars) and treated with z-VAD-fmk. Data are the means ± SD of triplicate determinations, each consisting of 150 cells. (C) Expression of TRAIL-R1 and TRAIL-R2 in various cell lines. Reverse transcriptase amplification of TRAIL-R1, TRAIL-R2, and actin from RNA of BJAB Burkitt lymphoma, 293T cells, 293T cells transfected with TRAIL-R1, and the Jurkat T lymphoma cell line. The control amplification was done in the absence of added cDNA. (D) BJAB cells transfected with dominant negative FADD (BJAB FADD-DN) or control transfectants (BJAB wt) were incubated with soluble FasL (sFasL) or sTRAIL for 16 hr at the indicated concentrations, and cell survival was measured by a cell proliferation assay. Immunity 1997 7, 831-836DOI: (10.1016/S1074-7613(00)80401-X)

Figure 3 Interaction of TRAIL-R1 and TRAIL-R2 with FADD and TRADD (A) (Top) 293T cells were transfected with the expression vectors coding for the cytoplasmic segment of the indicated receptors and an irrelevant plasmid to keep the total amount of plasmid constant. Lysates were immunoprecipitated with anti-Flag antibodies (precipitating the TRAIL receptors), and the presence of coprecipitating FADD and TRADD was assessed by Western blot analysis using anti-FADD and anti-Myc antibodies, respectively. IP, immunoprecipitating. (Bottom) The expression of the transfected proteins in the postnuclear cell extracts. (B) Analysis as in (A), but using Flag-tagged full-length TRAIL-R1 to demonstrate the interaction with FADD and TRADD. Immunity 1997 7, 831-836DOI: (10.1016/S1074-7613(00)80401-X)

Figure 4 Transcription Factor NF-κB Is Activated by TRAIL-R1 and TRAIL-R2 Effect of increasing TRAIL receptor expression on NF-κB activity in 293T cells, determined by a TNF promoter–derived, NF-κB–luciferase gene reporter assay. The control shows NF-κB activity induced by TRAMP. Data are the means ± SD of triplicate determinations. Immunity 1997 7, 831-836DOI: (10.1016/S1074-7613(00)80401-X)