Grx1 Activity Assay in Lung Tissue

Grx1 activity was assayed as described previously^{73 –75 (link)}. Briefly, lung tissue was lysed in 137 mM Tris-HCl, pH 8.0, 130 mM NaCl, and 1% NP-40. Lysates were then cleared by centrifugation and 100 μg was incubated with reaction mixtures containing Na/K phosphate buffer (0.1 mM, pH 7.5), 0.5 mM GSH (Sigma-Aldrich, G4251), 2 units/mL GSSG reductase (Sigma-Aldrich, G3664), 0.1 mM L-CySSG (Cayman, 17582), 0.2 mM NADPH (Roche, 10107824001) and 1.5 mM EDTA (pH 8.0) or with reaction buffer consisting of 137 mM Tris-HCl buffer (pH 8.0), 0.5 mM GSH (Sigma-Aldrich, G4251), 1.2 units GSSG reductase (Roche, 10105678001), 2.5 mM 2-hydroxyethyl disulfide (Sigma-Aldrich, 380474), 0.35 mM NADPH (Roche, 10107824001), 1.5 mM EDTA (pH 8.0). The reaction was prepared in a 96-well plate (final volume of 200 µL) and proceeded at 30 °C. The consumption of NAPDH was followed spectrophotometrically at 340 nm. In each sample, the spontaneous consumption of NADPH was subtracted. Data are expressed in units (U)/mg protein for lung homogenates in which 1 U equals the oxidation of 1 μmol NADPH/min.