BCAR3 promotes Rac1 activity.

<p>(<b>A</b>) BT549 cells transfected with siCtl (lane 1) or siB3-1 (lane 2) siRNA oligonucleotides were incubated for 72 hours, held in suspension for 90 minutes, then plated on 10 µg/ml fibronectin for 1 hour. GTP-bound Rac1 was isolated from whole cell lysates by incubation with PAK-1-binding domain agarose. Bound proteins (top panel) and total Rac1 (middle panel) were detected by immunoblotting with a Rac1 antibody, and BCAR3 knockdown was confirmed with a BCAR3-specific antibody (bottom panel). Quantification of the relative GTP-Rac1 level is shown. Data represent the mean ± SEM of 3 independent experiments (*, p<0.05). (<b>B</b>) BT549 cells were transfected with siCtl or siB3-1 oligonucleotides, incubated for 24 hours, followed by transfection with plasmids encoding Myc-RacL61 for an additional 48 hours. Cells were plated onto 10 µg/ml fibronectin-coated coverslips for 1–3 hours and processed for immunofluorescence as described in the methods. Actin is stained with Texas red-conjugated phalloidin (red) and Myc (RacL61) with fluorescein isothiocyanate (FITC) (green). Arrows indicate Myc-RacL61 expressing cells. Arrowheads indicate actin-rich stress fibers. The images shown are representative of 6 separate experiments. Scale bar = 15 µm. (<b>C</b>) The percentage of cells exhibiting actin-rich ruffles was determined for non-transfected and RacL61-expressing cells. Data represent the mean ± SEM of at least 36 cells per condition over to 2 separate experiments (*, p<0.05).