Horseradish peroxidase conjugated goat antimouse

Lung tissues were homogenized in ice-cold buffer containing 50 mmol/L Tris·HCl (pH 7.5), 1 mmol/L ethylene glycol tetraacetic acid, 1 mmol/L ethylenediaminetetraacetic acid, and a protease inhibitor cocktail (complete mini-tablets; Roche, Mannheim, Germany). Proteins (30 µg) were resolved on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions and electroblotted to a polyvinylidene difluoride membrane (Immobilon P, Millipore). After being blocked with 5% non-fat dry milk, the membranes were incubated with anti-GPX4 (1:750; SC-7269, Santa Cruz Biotechnology), anti-VEGF (1:750; SC-7269, Santa Cruz Biotechnology), and anti-β-actin (1:1000; C4 sc-47778, Santa Cruz Biotechnology) and then incubated with horseradish peroxidase-conjugated goat antimouse (Pierce Biotechnology, Rockford, IL, USA). Protein bands were detected using the BioSpectrum AC System from Pierce.

Lung tissues were homogenized in a cold buffer consisting of a mixture of protease inhibitors, 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L ethylene glycol tetraacetic acid, and 1 mmol/L ethylenediaminetetraacetic acid (entire mini tablets; Roche, Mannheim, Germany). A 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to resolve proteins (30 g) under reduced conditions, and the proteins were electroblotted to a polyvinylidene difluoride membrane (Immobilon P, Millipore). After being blocked with 5% non-fat dry milk, membranes were incubated with anti-β-actin (1:1000; C4 sc-47778, Santa Cruz Biotechnology) and anti-GPX4 (1:750; SC-7269, Santa Cruz Biotechnology), followed by horseradish peroxidase-conjugated goat anti-mouse (Pierce Biotechnology, Rockford, IL, USA). The protein bands were identified with a Pierce BioSpectrum AC System. The intensity of the TOMM20 and β-actin bands on AIDA was measured using densitometric analysis. After normalizing with β-actin, the densitometry unit of TOMM20 protein expression in the RA + NS group was designated as 1.