Fig. 4. (A) Effect of ARAP2 on cell-spreading rate. U118 cells (control), U118 cells expressing epitope-tagged ARAP2 (ARAP2) or U118 cells treated with siRNA to reduce expression of ARAP2 (siRNA) were trypsinized from plastic flasks, triturated and reseeded on coverslips coated with a solution of 10 µg/ml fibronectin. Cells were fixed at the indicated times and stained for paxillin and either endogenous ARAP2 or Flag epitope. The cells were then examined by confocal microscopy. To quantify the cell spreading at the indicated time, fluorescent images of 50 randomly selected cells were captured using 20x 1.4 NA objectives and surface areas were determined using ImagePro^® Plus 5.1 software package. Values are the mean ± standard error of the mean from two independent experiments. (B) Relative ARAP2 levels after siRNA treatment of U118 cells. Cells were either mock-transfected, or transfected with a control siRNA or siRNA targeting ARAP2 as indicated. Three days after transfection, the cells were lysed and analyzed by immunoblotting with specific antibodies to ARAP2.