Phospho enos s1177

Murine thoracic arteries were ground in liquid nitrogen and the resulting powder was lysed in RIPA-buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% (v/v) Igepal CA-630, 0.5% (w/v) deoxycholic acid, 0.1% (w/v) sodium dodecyl sulfate) for 30 min on ice. After removing cellular debris by centrifugation (16.000× g, 15 min, 4 °C), protein concentrations were measured using the Bradford reagent (BioRad, Munich, Germany). Endothelial cells were washed with PBS and scraped off the dishes on ice. After centrifugation, the resulting cell pellet was lysed in RIPA-buffer for 30 min on ice. After removing cellular debris by centrifugation (16.000× g, 15 min, 4 °C), protein concentrations were measured using the Bradford reagent (BioRad, Munich, Germany). Immunoblotting was performed with antibodies directed against eNOS (1:500) (Abcam, Cambridge, UK), phospho-eNOS (S1177) (1:500), phospho-eNOS (T495) (1:500) and the myc-tag (1:500) (all Cell Signaling Technology, Frankfurt, Germany). Blotting membranes were incubated with primary antibodies overnight at 4 °C before they were washed and incubated with secondary antibodies according to standard procedures. Detection was performed by enhanced chemiluminescence using the ECL reagent (GE Healthcare, Freiburg, Germany) and standard X-ray films. Semi-quantitative analyses were performed on scanned X-ray films using ImageJ 1.42q60 .