Fig. 2.

A: bovine aortic endothelial cells (ECs) were transfected with platelet endothelial cell adhesion molecule 1 (PECAM-1) short interfering RNA (siRNA), treated with hyperosmotic shock (HOS) or without hyperosmotic shock (No HOS) for 2 min (a condition known to induce PECAM-1 phosphorylation), and double-stained with anti-PECAM-1 (top) and anti-SHP-2 (bottom). The same field of view is shown for PECAM-1 and SHP-2. Top: several adjacent cells that were not transfected with the siRNA (thus expressing PECAM-1 at their shared cell border; arrows). When such cells were shocked with hyperosmotic medium (HOS), PECAM-1 became phosphorylated, which can be visualized by anti-SHP-2 staining (bottom left). Unshocked cells do not show cell border staining with anti-SHP-2 (right). These images demonstrate that SHP-2 relocalization can be used as a PECAM-1 phosphorylation reporter in ECs. Note that SHP-2 is normally localized throughout the cell. Data provided by Dr. Elena McBeath. B: SHP-2 relocalization to cell-cell contacts in confluent bovine aortic ECs. Anti-SHP-2 staining in unstimulated cells (static) show little cell border associated fluorescence. When ECs were exposed to 1 Pa of laminar shear stress for 5 min (flow), short linear anti-SHP-2 staining associated with cell border can be detected. Some of the stained areas are highlighted by straight lines (roughly 10 μm long). Note that many lines are roughly perpendicular to flow direction (arrow). Five minutes of hypersomotic shock (HOS), which is thought to cause membrane perturbation more equally throughout the entire cell border, causes extensive cell border staining, which surrounds the entire cell. Data provided by Brooke Krovic and Elena Mcbeath.