a, Effect of CFP-NS1 expression on EGF-stimulated ERK activation in HEK293 cells. CFP-NS1 and MYC-tagged ERK were co-expressed, and phosphorylation of MYC-tagged ERK was detected by Western blot with phosphospecific ERK antibodies. b, Cells transfected with the indicated oncogene along with CFP or CFP-NS1 were analyzed for ERK activation as in a. c, Effect of NS1 on AKT activation by RAS. Cells were transfected and analyzed as in b except HA-AKT was used in place of MYC-ERK. Quantification of results from b and c is provided in . d, NIH/3T3 transformation assay. Quantification of relative foci number for each oncogene is shown in the graphs. Results represent the ratio of foci number in presence of CFP-NS1 vs CFP alone and are the average of three independent experiments performed in triplicate +/− s.d. p values were determined by a Student’s t-test between CFP and CFP-NS1 for each oncogene. **, p<0.01, *, p<0.05. Effects of NS1 on oncogenic HER2/Neu, BRAF, and MEKK1 are shown in . e & f. Effects of NS1 on signaling and proliferation of human tumor cells. e. Titration of CFP-NS1 effects on ERK activation and proliferation (graph) in T24 bladder carcinoma cells (e) and A375 melanoma cells (f). Results in the graphs are the average of triplicate wells +/− s.e.m. shown by bars. Western blots in e and f are representative of at least 3 independent experiments. Full blot images for Figs 2a–c, e and f and , respectively.