Figure 3: Localization of tyrosine-phosphorylated proteins and vinculin in the renal arterial endothelial cells. En face preparations were stained with antiphosphotyrosine antibody (PY-20) (a–c) or antivinculin antibody (d–f) and observed by confocal laser scanning microscopy. The focal plane was adjusted to the surface (a and d), middle (b and e), or basal (c and f) portion of the endothelial cells. Tyrosine-phosphorylated proteins were observed along the apical plasma membrane in uniform staining (a). Strong staining for tyrosine-phosphorylated proteins was detected at sites of cell-to-cell apposition (b), with faint staining in the cytoplasm (c). Antivinculin staining was detected at sites of cell-to-cell apposition (e) and focal adhesion in the basal portion of the cells (f: arrowheads). No staining for vinculin was detected at the apical surface of the cells (d). The arrow indicates the direction of blood flow. Bar: 50 μm.