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

For double-labeling experiments to detect expression of both intracellular and cell surface fusion proteins, cells were fixed as described above, then treated with a rabbit antibody to v-sis, then with rhodamine-conjugated goat anti-rabbit antibody. The same cells were then permeabilized and treated with 10/~g/rnl of mAb P5D4 (Kries and Lodish, 1986) against the COOH terminus of VSV-G (kindly made available by William Balch, Scripps Clinic and Research Foundation, La Jolla, CA). These intracellular proteins were detected with a biotin-conjugated goat anti-mouse antibody which was in turn detected by FITC-conjugated streptavidin. For colocalization experiments, NIH3T3 cells grown on coverslips were infected with viral supernatants derived from sis-El-G-expressing cells, Coverslips were fixed and permeabilized as described above, and sis-E1-G was detected with a-sis antibody and rhodamine-conjugated goat anti-rabbit IgG, For colocaiization with the lectin, cells were then incubated with FITCconjugated Lens culinaris lectin (Sigma). For colocalization with mAb 10E6, cells were then incubated with 10E6 mAb, which was detected with FITC-conjngated goat anti-mouse IgG (kindly provided by V. Malhotra [Univ. California, Davis, CA] and W. J. Brown [Cornell Univ., Ithaca, NY)).