The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex

NIH3T3 cells were subjected to the infection protocol described above. 3 or 4 d after infection with viral supernatants, cells were incubated for 15 rain in MEM lacking cysteine and methionine. Each plate was labeled with 35 35 100/~Ci [ S]Met and [ S]Cys in 0.5 rnl MEM minus cysteine and methionine for 2 h. Calls were lysed with 1.0 rnl radioimmunnprecipitation assay buffer (10 mM sodium phosphate, pH 7.2, 150 mM NaC1, 1% NP-40, 1% DOC, 0.1% SDS, 10/~g/ml Aprotinin), clarified by centrifugation, and incuhated with a rabbit antiserum directed against bacterially synthesized v-sis protein, generously provided by Ray Sweet and Keith Deen (Smith, Kline and French, King of Prussia, PA) for 2 h at 4°C with rotation. Protein A-Sepharose beads (Sigma Chemical Co., St. Louis, MO) preincubated with unlabeled NIH3T3 cell lysate were used to isolate immune complexes. After separation on a sucrose gradient and extensive washing in radioimmunoprecipitation assay buffer, the beads were treated with 2× sample buffer, and immunoprecipitates were separated on a 15% SDS-PAGE gel and detected by fluorography. For analysis of dimer formation, half of each sample was treated with reducing sample buffer (50 mM Tris, pH 6.8, 2 % SDS, 20% 2-mercaptoethanol, 10% glycerol), while the other half was resuspended in nonreducing sample buffer which lacked the 2-mercaptoethanol. The samples were run on the same 15% SDS-PAGE gel and detected as described above.