Skip to main content
. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: Free Radic Biol Med. 2011 Sep 17;51(11):2108–2117. doi: 10.1016/j.freeradbiomed.2011.09.011

Figure 4. Free GSH and glutathionylated proteins (PSSG) in wild type (WT) and Grx2 knockout (KO) lens epithelial cells after H2O2 treatment.

Figure 4

(A) GSH level in WT and Grx2 KO lens epithelial cells (LECs). WT and Grx2 KO primary mouse LECs were treated with or without 100 μM H2O2 for 6 hrs. Cells were lysed in lysis buffer and GSH concentrations in cell lysates were determined using Ellman reagent described under “Material and methods”. Error bars indicate S.D., n=6, *P<0.05 vs. WT treated with H2O2. (B) PSSG levels in whole cell lysates in WT and Grx2 KO LECs. Wild type and Grx2 knockout were treated with or without 1 mM H2O2 for 30 min. Then the cells were lysed in lysis buffer and the total cell lysates were immunoblotted with anti-GSH (PSSG) antibody under non-reducing conditions. GAPDH was used as a loading control. The right panel depicts the relative pixel density of all the PSSG bands (see arrows) over GAPDH (with the untreated WT normalized to 1.0). Data presented are a typical representation of triplicate experiments. *P<0.05, vs. WT LECs. #P<0.05, vs WT LECs treated with 1 mM H2O2 for 30 min. (C) PSSG levels in mitochondrial fraction of WT and Grx2 KO cells. Cells in (B) were used for mitochondrial isolation and the mitochondrial extracts were used for Western blot (WB) detection for PSSG. Mitochondrial protein VDAC was used as a loading control. The bottom panel depicts the relative pixel density of all the PSSG bands (see arrows) over VDAC (with the untreated WT normalized to 1.0). Data presented are a typical representation of triplicate experiments. *P<0.05, vs. WT LECs. #P<0.05, vs WT LECs treated with 1 mM H2O2 for 30 min. (D) Deglutathionylation of mitochondrial PSSG with imported Grx2. Grx2 KO cells were imported with 20 μM purified wild type Grx2 recombinant protein (rGrx2), Grx2-C70S/C73S double mutated protein (Mutant). After 4 hours of incubation, the cells were treated with 1 mM H2O2 for 30 min. After treatment, the mitochondrial fraction of each group was analyzed for PSSG. His-Tag immunoblotting was done to verify the delivery of recombinant Grx2 protein. The bottom panel depicts the relative pixel density of all the PSSG bands (see arrows) over VDAC (with the KO LECs without import normalized to 1.0). Data presented are a typical representation of triplicate experiments. *P<0.05, vs. KO LECs treated with 1 mM H2O2 for 30 min.