Chemiluminescence detection kit

Analysis of oxidized proteins was performed by western blot in liver homogenates using an Oxyblot kit (Millipore Bioscience Research Reagents, Temecula, CA). The same amounts of liver proteins (35 μg) were reacted with dinitrophenylhydrazine (DNP) for 20 min, followed by neutralization with a solution containing glycerol and 2-mercaptoethanol, resolved in 12.5% sodium dodecyl sulphate–polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, blocked with nonfat milk, and incubated with a rabbit anti-DNP antibody (1: 150) at 4 °C overnight. After washing, the membrane was incubated with the secondary antibody (1:300) conjugated to horseradish peroxidase and detected by a chemiluminescence detection kit (Cell Signaling Technology Inc., Danvers, MA). Reactive bands were visualized by the enhanced chemiluminescence method on a VersaDoc Image System (Bio-Rad Laboratories, Hercules, CA).

Proteins were prepared from HASMCs or arteries. For western blot analysis, proteins were electrophoresed on 10% SDS–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Each membrane was blocked with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween 20 (TBS-T) for 1 h and then incubated with the primary antibody at 4 °C overnight. The membrane was washed and incubated with the secondary antibody conjugated with horseradish peroxidase in 5% non-fat milk in TBS-T buffer for 1 h. Detection was carried out using a Chemiluminescence Detection Kit (Cell Signaling Technology)50 (link)51 (link). Uncropped images of blots are presented in Supplementary Fig. 20.

The analysis of oxidized proteins was performed by western blot in liver mitochondria using an Oxyblot kit (Millipore Bioscience Research Reagents, Temecula, CA, USA) [16 (link)]. The same amounts of mitochondrial proteins (35 µg) were reacted with dinitrophenyl hydrazine (DNPH) for 20 min, followed by neutralization with a solution containing glycerol and 2-mercaptoethanol, resolved in 12% SDS-polyacrylamide gel electrophoresis. After the transfer to a nitrocellulose membrane, a blocking step with non-fat milk and incubation with a rabbit anti-DNPH antibody (1: 150) at 4 °C overnight followed. After washing, the membrane was incubated with the secondary antibody (1:300) conjugated to horseradish peroxidase and detected by a chemiluminescence detection kit (Cell Signaling Technology Inc., Danvers, MA, USA). Reactive bands were visualized by the enhanced chemiluminescence method on a VersaDoc Image System (Bio-Rad Laboratories, Hercules, CA, USA). Band density was determined with TotalLab software. The test provides a qualitative analysis of the total proteins’ oxidation state change.