Fig. 5.

(A) Expression of the DmVav mutant proteins used in these experiments. Expression vectors encoding the indicated mutants of Myc-tagged DmVav (top) were transfected in COS1 cells. 24 h after transfection, total cell lysates were obtained and analyzed by immunoblot analysis with anti-Myc antibodies. The migration of DmVav proteins is indicated with an arrow. (B) Transforming activity of DmVav (Δ1–207) and mouse Vav (Δ1–186) proteins with inactivating point mutations in the DH, PH, ZF, SH2, and SH3 domains. Focus assays were performed as indicated in the legend to Fig. 1. ΔN, N-terminal deleted forms of DmVav and mouse Vav; mut, mutant. (C) Morphological change induced by DmVav and mouse Vav mutant proteins in NIH3T3 cells. Cells were transfected with plasmids encoding the indicated mutant proteins (left side) of mouse Vav (Δ1–186) (panels A–U) and DmVav (Δ1–207) (panels A′–R′). After transfection, cells were stained with rhodamine-phalloidin and with antibodies to the Vav DH region (panels A–U) or the Myc epitope (panels A′–R′). After incubation with FITC-labeled secondary antibodies, cells were subjected to confocal immunofluorescence analysis. The localization of F-actin and Vav proteins is shown in red and green, respectively. The areas of co-localization are shown in yellow. DH-PH-ZF (lower panel) refers to a truncated version of mouse Vav containing only the central DH-PH-ZF regions. The specific mutations used to inactivate each domains have been indicated in (A) and the main text.