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

For infections, NIH3T3 ceils were transfected as described above with 9/~g of MLV DNA plus 1/~g pZAP helper virus. Transfected cells were split as described above, and allowed to grow for 4 d without refeeding. The supernatant media, containing viral particles, were then collected, centrifuged to pellet any cells, and used to infect monolayers of fresh NIH3T3 cells, split 1-2 x 10 s cells per 60-ram plate one day earlier. Polybrene (4 #g/ml) was added to the newly infected cells to increase the efficiency of infection. Infected cells were refed the following day with fresh DME media, and were used 2 or 3 d later for labeling and intmunoprecipitations or immunofluorescence. This protocol results in a very high percentage of cells expressing the desired protein.