Enhanced horseradish peroxidase luminol chemiluminescence reagent

Protein samples (30 µg each) were separated on a Bis-Tris gel and transferred to a nitrocellulose membrane. After the transfer to a nitrocellulose membrane, the blots were blocked in 5% milk powder in TBST (10 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl with 0.1% Tween 20) for one hour at room temperature. The primary antibodies were diluted in the same solution at 4 °C over night. The following primary antibodies were used: rabbit monoclonal antibody against phospho-eNOS at Ser1177 (catalog number 9571, Cell Signaling; 1:1000), mouse monoclonal antibody against eNOS (catalog number 610297, BD Bioscience; 1:2000), rabbit monoclonal antibody against phospho-Akt Ser473 (catalog number 4060, Cell Signaling; 1:2000), rabbit monoclonal antibody against Akt (catalog number 4691, Cell Signaling; 1:1000), mouse monoclonal antibody against β tubulin I (catalog number T8328, Sigma Aldrich; 1:5000). Subsequently, blots were washed with TBST and incubated for one hour with a horseradish peroxidase-conjugated secondary antibody diluted in 5% milk in TBST. After washing in TBST and TBS, the immunocomplexes were visualized using an enhanced horseradish peroxidase/luminol chemiluminescence reagent (PerkinElmer Life and Analytical Sciences, Boston, MA) according to the manufacturer’s instructions. Densitometric analysis of scanned blots was performed using the Quantity One software (Bio-Rad).

Western blot analyses were performed with total protein samples (30 µg each) from the aorta. The following primary antibodies were used: mouse monoclonal antibody against eNOS (catalog number 610297, BD Transduction Laboratories, Franklin Lakes, NJ, USA; 1:2000), SOD2 (ADI-SOD-110, Enzo Life Sciences, New York, NY, USA; 1:2000). The Western blot was performed as previously described [33 (link),34 (link)]. The protein samples were separated on a Bis-Tris gel and transferred to a nitrocellulose membrane. The blots were blocked in 5% milk powder in TBST (10 mM Tris-HCl, pH 7.4, 150 mM NaCl with 0.1% Tween 20) for one hour at room temperature. The primary antibodies were diluted in the same solution used for blocking at 4 °C overnight. The blots were then washed in TBST and incubated with a horseradish peroxidase-conjugated secondary antibody diluted in 5% milk in TBST for one hour. After washing in TBST and then in TBS, the immunocomplexes were visualized using an enhanced horseradish peroxidase/luminol chemiluminescence reagent (PerkinElmer Life and Analytical Sciences, Boston, MA, USA), according to the manufacturer’s instructions. A densitometric analysis of scanned blots was performed using Quantity One software (Bio-Rad, Feldkirchen, Germany).