Preparation of LEC protein extracts and Western blot analysis.

Preparation of protein from LECs for Western blot analysis was performed as previously described (5, 42, 64). Cell lysates from third- to sixth-passage LECs derived from control and shunt lambs were used. Protein concentration in each sample was quantified using a Nano Drop Spectrophotometer (ND-1000; Thermo Fisher Scientific, Waltham, MA). For Western blot analysis, 20 μg total protein were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were then blocked with 5% nonfat dried milk in 130 mM NaCl and 25 mM Tris (TBS, pH 7.5) for 1 h at room temperature. Membranes were blocked and subsequently exposed to primary antibodies against endothelial nitric oxide synthase (eNOS; Santa Cruz), inducible nitric oxide synthase (iNOS; Santa Cruz), neuronal nitric oxide synthase (nNOS; Santa Cruz), LYVE-1 (Abcam), Prox1 (EMD Millipore), 90-kDa heat shock protein (HSP90) (BD Transduction Laboratories), caveolin-1 (Santa Cruz), calmodulin (Santa Cruz), phospho-eNOS-serine-1177 (Cell Signaling), or nitrotyrosine (CalBiochem), as well as β-actin (Abcam), which served as a loading control. Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibodies, chemiluminescence was then used to detect bands (SuperSignal West Pico Chemiluminescent Substrate kit; Pierce Biotechnology, Rockford, IL). Densitometry was performed using a public domain Java image-processing program, Image J (NIH Image).