Tgx minigels

Fresh human arterioles were homogenized in ice-cold lysis buffer containing 25 mM Tris-HCL (pH 7.4), 150 mM NaCl, 0.1% SDS, and 1% NP-40, supplemented with protease and phosphatase inhibitors, and centrifuged at 12,000 g for 10 min at 4°C. Protein concentrations of the supernatants were measured by a BCA kit according to the manufacturer’s instructions. Samples were aliquoted and frozen in liquid N₂, stored at −80°C before use. Protein samples (20 μg) were separated by SDS-PAGE on 10% Bio-Rad TGX mini gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked by 5% non-fat dry milk (NFDM) or bovine serum albumin (BSA) for 1 hour at room temperature and incubated overnight at 4 °C with a primary monoclonal antibody against NADPH oxidase 4 (kindly given by Dr. Doroshow at the NIH/NCI) (1:500 dilution in TBST) in TBST with 5% NFDM, or a monoclonal antibody against β-actin (1:40,000 dilution in TBST) in TBST with 2% NFDM. Blots were washed with TBST and then incubated with HRP-conjugated secondary antibody (1:20,000 dilution in TBST with 2% NFDM) for 1 hour at room temperature. Membranes were developed using ECL prime reagent and KONICA developer. The films were scanned using an EPSON scanner.