Sulfobiotics protein redox state monitoring kit plus

HPASMCs (ScienCell Research Laboratories, Carlsbad, CA, USA) were serum-starved overnight and treated with recombinant human PDGF-BB or H
₂O
₂ for 10, 15 or 30 min. Protein thiol states were monitored using SulfoBiotics Protein Redox State Monitoring Kit Plus (Dojindo Molecular Technologies, Rockville, MD, USA) in accordance with the manufacturer’s instructions. Briefly, cells were washed, proteins precipitated with trichloroacetic acid and “Protein-SHifters” were added to each sample. Samples were then loaded onto a sodium dodecyl sulfate polyacrylamide gel and electrophoresed. The gel was exposed to UV light to cut the “Protein-SHifters.” The resultant non-reducing SDS polyacrylamide gel was electroblotted to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% milk for 30 min at room temperature and incubated with the anti-Prx6 antibody produced in rabbit (Sigma-Aldrich Chemical Company, St. Louis, MO, USA; Catalogue no. P0058; 1:1,000 dilution) at 4°C overnight. The membrane was then washed three times and incubated with goat anti-rabbit IgG-horseradish peroxidase conjugate (Bio-Rad; Catalogue no. 1706515; 1:3,000 dilution) for 45 min at room temperature. After washing three times, signals were obtained using an Enhanced Chemiluminescence System (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA).

Protein thiol redox states were monitored using the -SulfoBiotics- Protein Redox State Monitoring Kit Plus (Catalog # SB12; Dojindo Molecular Technologies). Samples were labeled with the Protein-SHifter Plus in accordance with the manufacturer’s instructions. After cell extracts were subjected to SDS-PAGE, gels were exposed to the UV light on a transilluminator to remove Protein-SHifter. Proteins in the gel were then electro-transferred to the Immobilon-FL Transfer Membrane. The membrane was blocked with Odyssey Blocking Buffer for one hour at 25°C and incubated overnight with the rabbit anti-Prx6 antibody (Catalog # P0058; MilliporeSigma) at 4°C. Washed membranes were then incubated with anti-rabbit IRDye 680RD for one hour at 25°C in the dark. Signals were then captured by using the Odyssey Infrared Imaging System, and band intensities were analyzed by densitometry.