Phosphorylated enos ser1177

Protein levels of pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β and protein levels of eNOS and phosphorylated eNOS (Ser1177) in the aortic tissue were analyzed by western blot. Briefly, the aortic tissue was homogenized in a mammalian tissue lysis buffer with protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was assessed by the Bradford method. Samples were loaded in a 12% SDS-polyacrylamide gels and then transferred to a polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). After blocking for 1 h in 5% non-fat dry milk, the membranes were immunoblotted with primary antibodies to TNF-α, IL-1β (Cell Signaling Technology, Beverly, MA, USA), eNOS (BD Biosciences, San Jose, CA, USA), phosphorylated eNOS Ser1177 (BD Biosciences, San Jose, CA, USA) and β-actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) overnight at 4°C. The membranes were washed and then incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) at room temperature for 1 h. Membranes were incubated with the enhanced chemiluminescence detection reagents (Amersham, Arlington Heights, IL, USA) and band densities were analyzed with ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). All data were expressed as relative units normalized to β-actin.

An equal amount of protein was loaded on SDS-PAGE gel and transferred to PVDF membranes (GE Healthcare, Buckinghamshire, UK), and blocked for 1 hour at room temperature in 5% BSA or skim milk in Tris-buffered saline with Tween 20 (TBST). The primary antibodies were used as follows; VEGF (1000:1, Santa Cruz, sc-152), phosphorylated-eNOS (Ser1177) (1000:1, BD, #612393), total-eNOS (1000:1, Enzo, ADI-905-386), phosphorylated-ERK1/2 (Thr202/Tyr204) (1000:1, Cell Signaling, #9101), total-ERK1/2(1000:1, Cell Signaling, #9102), phosphorylated-protein kinase B (Akt) (Ser473) (1000:1, Cell Signaling, #4060), total-Akt (1000:1, Cell Signaling, #9272), CD31 (1000:1, Abcam, ab32457), HGF (500:1, abcam, ab83760), basic FGF (500:1, Santa Cruz, sc-79) and α-tubulin (1000:1, Sigma, T9026). The regions containing protein were visualized by the enhanced chemiluminescence system (ECL Prime Western Blotting Detection Regent, GE Healthcare, Buckinghamshire, UK). Densitometric analysis was performed by the Image J Software (NIH, USA).