Anti phospho enos antibody

Anti-phospho-eNOS antibody was purchased from Cell Signaling (Beverly, MA, USA). Anti-NOS3, anti-β-actin, anti-phospho-Akt and total Akt antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed by boiling 30 µg of whole cell lysate or 30 µg of tissue homogenate (obtained from rat aorta) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer, before separation by electrophoresis and transfer to a nitrocellulose membrane. After incubation in appropriate primary and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), chemiluminescent signaling was developed using Super Signal West Pico or Femto Substrate from Thermo Fisher Scientific (Pierce, Rockford, IL, USA). Blots were imaged and band densities quantified with a Gel Doc 2000 Chemi Doc system using Quantity One software from Bio-Rad (Hercules, CA, USA). Values were normalized to a β-actin loading control.

Anti-phospho-eNOS antibody was purchased from Cell Signaling (Beverly, MA, USA). Anti-NOS3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blotting analysis was performed by adding 30 μg tissue homogenate (obtained from rat aorta) to SDS–PAGE loading buffer followed by boiling and separation by electrophoresis and transfer onto nitrocellulose membranes. After incubation with appropriate primary and peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), the chemiluminescent signal was developed using Super Signal West Pico or Femto Substrate from Thermo Fisher Scientific (Rockford, IL, USA). Blots were imaged and band densities quantified with a Gel Doc 2000 Chemi Doc system using Quantity One software (Bio-Rad, Hercules, CA, USA). Values were normalized relative to β-actin loading control.