Figure 4

Vilse and Robo1 interact with Robo4 and Rho GTPase homeostasis is important for Robo4 mediated directional migration. A shows the interaction between Robo4 and Vilse in 293 cells. Myc-Vilse and V5-tagged human Robo4 were co-transfected into 293 cells in different combinations as indicated by + and -. Slit2N was added to the transfected cells and lysates were immunoprecipitated by myc antibody followed by western with V5 antibody. B shows IP/Western analysis for rat Robo1 and human Robo4 (left panel) or rat Robo1 and mouse Robo4 (right panel). The antibodies are indicated to the left and right of the gels. Tub: tubulin, mR4: mouse Robo4. The top gel in each panel represents the IP'd lysate western blotted with the indicated antibody. The rest of the gels are western blots for the respective proteins either present (+) or absent (-) as indicated for each sample in the top of the gel. C shows pull down analysis for Cdc42-GTP and Rac-GTP in Con (control) siRNA, robo4 (R4) siRNA and robo4 siRNA plus zfRobo4 transfected endothelial cells. Quantitation of the western blots was performed as described before [22]. Antibodies used for westerns are shown to the left of the blot. D shows western blots with myc and actin antibody of lysates from control lacZ siRNA (lane 1) or robo4 siRNA (lane 2) co-transfected with myc-IRSp53 constructs in endothelial cells. E and F are confocal images of F-actin phalloidin (red) stained endothelial cells transfected with reagents shown below the panel. Slides were mounted in media containing DAPI (blue), which stains nuclei. For A, B, C and D + indicates addition of the reagent on the left, IP-immunoprecipitation, WB-western blot, antibodies indicated on right, For panels C, D, E and F Con: control lacZ siRNA, R4: robo4 siRNA.