Fig. 2.

Transforming activity of DmVav proteins with Y to F mutations in the Ac region. (A) Focus formation assays were conducted with plasmids (1 μg each) encoding DmVav or mouse Vav that were either wild type (WT) or mutated in the indicated tyrosine residues. Y3xF is an abbreviation for the triple Vav Y142F + Y160F + Y174F and DmVav Y165F + Y183F + Y194F mutants. As a comparative control, we included transfections with a plasmid encoding DmVav (Δ1–207; 0.1 μg). 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 represented in a bar histogram. Values were normalized considering the amount of plasmid DNA used in each transfection. (C) 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.