Figure 6.

MC integrin β8 and GEC PECAM-1 are synergistically cytoprotective. A: Immunoblots from whole cell lysates (20 μg total protein) from mouse glomerular endothelial cells (GEC), kidney endothelial cells (KEC) from wild-type and PECAM-1^−/− mice, and GEC transfected with adenovirus (Ad) or PECAM-1 adenovirus (AdP). The PECAM-1 band appears as a doublet due either to the presence of more than one isoform⁷⁶ or glycosylated and unglycosylated proteins.⁷⁷ Blots were stripped and re-probed with anti-tubulin antibodies as a loading control (lower panel). B: Untransfected (mGEC), adenoviral vector-transfected (Ad), and PECAM-1 adenoviral vector-transfected (AdP) mouse GEC were incubated for 24 hours with serum-free DMEM, followed by conditioned media obtained from quiescent wild-type MC (+/+), Itgb8^−/− (−/−) MC or DMEM (D) only (16 hours, 37°C), and then evaluated for apoptosis by annexin V labeling. *P < 0.01 compared to D- and +/+ treated groups. C: Conditionally immortalized endothelial cells derived from PECAM-1^−/− or PECAM-1^+/+ mouse kidneys were grown to near confluence in complete media under permissive 31°C conditions, and then placed in serum-free DMEM (72 hours, 37°C). Cells were then washed with Hanks' solution, and incubated with conditioned media from wild-type (+/+) or Itgb8^−/− (−/−) MC (8 hours, 37°C) and then evaluated for apoptotic nuclear morphology by DAPI labeling by two observers blinded to experimental conditions. *P < 0.01 compared to other groups.