FIGURE 2.

Ang-2 binding to α5β1 integrin. A, CHO cells were cotransfected with integrin cDNAs and GFP cDNA. At 24 h after transfection, the cells were detached, incubated with FLAG-tagged Ang-2, and probed by an anti-FLAG antibody conjugated to phycoerythrin. Fluorescence intensities were analyzed by flow cytometry. Levels of GFP expression (y axis) and Ang-2 binding (x axis) are depicted as dot plots. Geometric mean values of Ang-2 binding in cells expressing different levels of GFP are plotted as larger red dots. B, levels of Ang-2 binding (y axis) and GFP expression (x axis) in A were plotted as line graphs using the mean values of Ang-2 binding in each range of GFP expression. C, FLAG-Ang-2 (10 μg/ml) was incubated in plates coated with 5 μg/ml integrin α5β1, 5 μg/ml Tie2, or BSA. Bound FLAG-Ang-2 was detected using an anti-FLAG antibody conjugated to HRP. The optical density (OD) at 450 nm is plotted as a bar graph. Error bars, S.D. (*, p < 0.001). D, a quantitative ELISA was performed using a series of diluted concentrations of Ang-2. E–G, U87MG cells were transfected with siRNAs targeting α5 or β1 integrin. Reduced expression of integrins on the cell surface was confirmed by flow cytometric analysis of cells transfected with siRNA targeting α5 integrin (E) and β1 integrin (F). G, Ang-2 binding to cells transfected with siRNA targeting integrin α5 or β1 was analyzed by flow cytometry. Mean fluorescence intensities of siRNA-transfected cells were normalized to that of the control siRNA-transfected group. Data are shown as mean ± S.D. (error bars) (*, p < 0.05; **, p < 0.01).