Figure 8.

R-Ras is activated at the leading edge and upon adhesion to collagen. (A) T47D control cells and cells expressing activated R-Ras (38V) were plated on collagen-coated coverslips and immunostained for R-Ras, which localizes to membranes (right panel). Control motile cells also localize endogenous R-Ras to the leading edge (see arrows in left panel). Scale bar, 25 μm. (B) Cell lysates from pseudopodia and cell bodies were prepared as previously described (Cho and Klemke, 2002). Raf RBD:GST, 50 μg, was used to pulldown GTP-bound R-Ras from lysates as detailed in the Materials and Methods. ERK was also analyzed as a loading control (Brahmbhatt and Klemke, 2003). R-Ras activity is significantly increased (^*p < 0.05 vs. cell body) in the protruding pseudopod. (C) T47D cells were plated on collagen or BSA-coated plates and the R-Ras activity assay performed. Cells plated on collagen showed increased (p = 0.12 vs. no collagen) R-Ras activity. All quantification was performed on three individual experiments.