FIGURE 7.

Detection of glutathionylated proteins (PSSG) in the lenses of WT and Grx2 KO mice. A, comparison of protein glutathionylation in the lens of WT and Grx2 KO mice. Soluble fraction of lens homogenates from WT and Grx2 KO mice (1, 7, and 16 months old) were analyzed by Western blotting using anti-PSSG antibody under nonreducing conditions. GAPDH was used as a loading control. The graph on the right depicts the relative pixel density of all the PSSG bands (indicated by arrows) over GAPDH (with the 1-montg-old WT normalized to 1.0). B, S-glutathionylated protein identification. Whole lens lysate from 16-month-old Grx2 KO mice was immunoprecipitated for glutathionylated proteins using anti-PSSG antibody and then was separated with SDS-polyacrylamide gel followed by Coomassie Blue staining. Proteins showing positive immunoreactivity (indicated by arrows) with anti-PSSG antibody were cut and identified by liquid chromatography-tandem mass spectrometry as predominantly actin, αA-crystallin, and βB2-crystallin. For confirmation, glutathionylated proteins were immunoprecipitated (IP) by using anti-PSSG antibodies, followed by immunoblot (IB) detection of actin (C), βB2-crystallin (D), and αA-crystallin (E). Blots labeled “IB: actin,” “IB: αA-crystallin”, and “IB: βB2-crystallin” are control Western blots for actin, βB2-crystallin, and αA-crystalline, respectively. The right panels show the pixel density graphs of glutathionylated actin, βB2-crystallin, or αA-crystalline in comparison with the 1-month-old WT lens, respectively. Data represent the means ± S.D. of three independent experiments. *, p < 0.05 versus age-matched WT; **, p < 0.01 versus age-matched WT.