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

NIH3T3 cells were maintained at 370C, 10% COz in DME containing 10% calf serum, fed every 3 d with fresh medium, and passaged when 70-80% confluent. For focus assays, cells were split at a density of 2 x 10 s cells per 60-ram plate and transfected the following day with 50 ng of expressing plasmid, 50 ng pZAP helper virus (Hoffman et al., 1982) , and 9.9 t~g carrier plasmid DNA using the calcium phosphate precipitation protocol (Chen and Okayanm, 1987) . Cells were incubated with precipitate for 18-20 h at 37°C, 3% CO2, then refed and transferred to 10% CO2. The cells from each 60-mm plate were split 1:12 the following day and scored for foci 4-5 d later.