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note
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DR4 and DR4Δ (amino acids 1 to 351) were cloned into pCMV1FLAG (IBI Kodak). The constructs encoding TNFR-1 and TNFR-1Δ have been described (15). The cDNAs encoding the extracellular domains of DR4 (amino acids 87 to 217) and TNFR-1 (amino acids 37 to 205) were obtained by polymerase chain reaction (PCR) and cloned into a modified pCMV1FLAG vector that allowed for in-frame fusion with the Fc portion of human immunoglobulin G. cDNAs encoding soluble TRAIL (amino acids 95 to 281) and soluble TNF-α (amino acids 77 to 233) were obtained by PCR and subcloned into pCMV1FLAG and pSectagB (Invitrogen), respectively.
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Cell death assays were performed as described (6). MCF7 cells were transfected by using the lipofectamine procedure (BRL) according to the manufacturer's instructions. The 293 cells were transfected by means of calcium phosphate precipitation.
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In vivo interaction and NF-κB luciferase assays were done as described (10).
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1842269979
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note
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We transfected 293 cells with constructs encoding either receptor-Fc chimeras or ligands, and the conditioned media was harvested 72 to 80 hours later, clarified by centrifugation, divided, and stored at -80°C. For binding assays, equal amounts of receptor-Fc- and liqand-containing conditioned media were mixed in buffer containing 50 mM Hepes, pH 7.0, 150 mM NaCl, 1 mM EDTA, 0.5 % NP-40, and a protease inhibitor mixture and the sample was incubated at 4°C with continuous rotation for 4 hours. Receptor-Fc-ligand complexes were precipitated with protein G-Sepharose, extensively washed with the above buffer, boiled in SDS sample buffer and resolved on a 12% SDS-polyacrylamide gel. Bound ligands were identified by immunoblotting.
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A PCR fragment encoding soluble FLAG-TRAIL (amino acids 95 to 281) was cloned into the His-tag vector pET15b (Novagen). The His-FLAG-TRAIL was purified by nickel chelate affinity chromatography according to the manufacturer's instructions.
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note
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We thank S. Nagata for the Fas-Fc and soluble FasL expression constructs, J. Bretz and other members of the Dixit lab for helpful discussions, M. Garg for preparing His-FLAG-TRAIL protein, I. Jones for assistance in preparing the figures, and J. DeJohn for secretarial help. Supported by NIH grants ES08111 and DAMD17-96-1-6085.
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