Figure 3.

BCAR3 overexpression in MCF-7 and T47D cells increases BCAR3-p130^Cas interactions and promotes cell migration. A, MCF-7 and T47D cells were transiently transfected with vector (lanes 1, 3, 5, and 7) or BCAR3-encoding plasmids (lanes 2, 4, 6, and 8). Twenty micrograms of total cell lysate (lanes 1, 2, 5, and 6) or p130^Cas immune complexes generated from 200 μg of total protein (lanes 3, 4, 7, and 8) were immunoblotted for p130^Cas (top) or BCAR3 (bottom). B, MCF-7 and T47D cells were cotransfected with plasmids encoding GFP plus vector or BCAR3. Twenty-four hours later, cells were seeded onto Boyden chambers and allowed to migrate toward serum for 24 h at 37°C (see Materials and Methods for calculation of relative migratory index). Columns, mean for five or seven independent experiments, respectively; bars, SD. Black columns, vector-transfected cells; gray columns, BCAR3-transfected cells. *, P < 0.05 relative to vector-transfected cells. C, T47D cells transfected with BCAR3-expressing plasmids were plated onto fibronectin-coated coverslips 24 h posttransfection and processed for immunofluorescence 24 h later. BCAR3 and p130^Cas were visualized by epifluorescence microscopy as described in Materials and Methods. Overexpressed BCAR3 colocalized with p130^Cas at the periphery of the cell (arrowheads).