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

NIH3T3 cells were grown on coverslips and transfected as described above with 10 t~g of Rous sarcoma virus constructs, or subjected to infection with viral supernatants (see above). To detect intracellniar v-sis fusion proteins, ceils were fixed in 3 % paraformaldehyde/PBS for 10 rnin, followed by permeabilization in 1% Triton/PBS for 5 rain. Cells were then incubated with a rabbit antiserum directed against the v-sis protein, followed by a rhodamine-conjugated goat anti-rabbit antibody. To detect cell surface v-sis fusion proteins, cells were fixed with paraformaldehyde and incubated with antibodies without permeabilization, as described previously (Hannink and Donoghue, 1986a; Lee and Donoghue, 1992) .