Fig. 1.

Transforming activity of DmVav proteins with amino acid deletions in the CH-Ac region. (A) Focus formation assays were conducted with plasmids encoding DmVav (upper row), mouse Vav (middle row), or mouse Vav2 (lower row) that were either wild type (WT) or deleted in the indicated N-terminal regions. NIH3T3 cells were transfected with vectors encoding either wild type versions (1 μg each) or mutant Vav proteins (0.1 μg each). As a control, we used cells transfected with high molecular weight calf thymus DNA alone (None). After transfection, cells were cultured for 15 days and stained with Giemsa to visualize the foci of transformed cells. (B) Transforming activity of Vav mutants. Foci obtained in the above transfections were counted de visu and numbers obtained represented in a bar histogram. Values were normalized considering the amount of plasmid DNA used in each transfection. (C) Morphology of the foci obtained with the indicated oncogenes. (D) Expression of the DmVav proteins used in these experiments. COS1 cells were transfected with either empty plasmid (Mock) or with expression vectors encoding the indicated DmVav proteins. After 48 h, total cell extracts were obtained and protein expression evaluated by anti-Myc immunoblots (upper panel). Equal loading of samples was demonstrated using anti-γ-tubulin antibodies (lower panel). The migration of molecular weigh markers is indicated on the right. The position of DmVav and γ-tubulin proteins is indicated by arrows on the left. S, supernatant fraction after the centrifugation of cellular extracts after cell lysis. P, pellet obtained after the centrifugation of the cellular lysates. WB, Western blot.