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84920308223
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note
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The plasmid Gal4BD-DR3, which encodes the GAL4 DNA-binding domain fused to the intracellular domain (amino acids 227 to 418) of DR3, was used as bait in a yeast two-hybrid screen of a HeLa cell cDNA library (Clontech). The isolated positive clones were analyzed as described (3). Full-length SODD cDNA was obtained by screening a λZAP human Jurkat T cell cDNA library by standard methods (3).
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84920308221
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note
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For protein-protein interaction assays, subconfluent 10-cm dish cultures of 293 cells were transfected by the calcium phosphate method (3). To determine SODD interaction with DDRs, we used expression vectors encoding Flag-SOOD and CST-DDRs or GST. Twenty-four hours after transfection, cells were washed in phosphate-buffered saline and lysed in E1A lysis buffer (3). Lysates were precipitated with glutathione agarose beads (Pharmacia Biotechnology), and the beads were washed three times with E1A buffer and twice with E1A buffer containing 1 M NaCl. The precipitates were fractionated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane. The coprecipitated SODD was detected by immunoblot analysis with polyclonal antibodies to the Flag epitope (Santa Cruz Biotechnology). To determine SODD binding to various TNF-R1 mutants (2, 3), we incubated cell lysates for 2 to 4 hours at 4°C with monoclonal antibody 985 to TNFR1 (5) or control mouse immunoglobulin G (IgC) monoclonal antibody (Sigma) and 25 μl of 1:1 slurry of protein G-Sepharose (Pharmacia). Beads were washed twice with 1 ml of E1A buffer, twice with 1 ml of high salt E1A buffer, and twice again with E1A buffer before immunoblot analysis was performed.
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10544236916
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Marsters, S.1
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84920308219
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GenBank accession numbers AA319013, AA362082, H10621, and T33545
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GenBank accession numbers AA319013, AA362082, H10621, and T33545.
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84920308218
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note
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Rabbit polyclonal antisera were generated against a SODO peptide (amino acids 292 to 313) and against purified His-tagged SODD produced in Escherichia coli. The upper band of the protein doublet in Fig. 1C is presumably phosphorylated SODD. This band predominates in cell lines that express low levels of SODD, whereas both bands are apparent in cell lines that express higher levels (12).
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25
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0030611595
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J. D. Woronicz, X. Gao, Z. Cao, M. Rothe, D. V. Goeddel, Science 278, 866 (1997).
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Woronicz, J.D.1
Gao, X.2
Cao, Z.3
Rothe, M.4
Goeddel, D.V.5
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84920308217
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note
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We thank L Huang for synthesizing the SODD peptide for antibody generation, V. Dixit for providing the DR3 cDNA, A. Ashkenazi for providing DR4 and DR5 cDNAs, D. Baltimore for supplying the parental mammalian cell expression vector for GST, pEBG, and L. Medin for help with the figures.
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