The relationship between the amount of S-glutathionylation and the vicinal dithiols-containing molecules.

<p>HEK 293 cells were pretreated with 1 µM PAO for 30 minutes before treating with 0.25 mM diamide or 0.5 mM hydrogen peroxide. The preparation of PAO was described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004015#s3" target="_blank">Methods</a>, and the effect of PAO on cellular glutathione was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004015#pone-0004015-t003" target="_blank">Table 3</a>. Cell lysates were prepared, and modified proteins were detected as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004015#s3" target="_blank">Methods</a>.</p

The Effect of PAO (1.0 µM) on Glutathione Concentration.

<p>The Effect of PAO (1.0 µM) on Glutathione Concentration.</p

Protein S-glutathionylation in HEK 293 cells.

<p>(A) The pattern of overall S-glutathionylated proteins is shown in a time course experiment with 0.5 mM diamide or 0.5 mM hydrogen peroxide-treated HEK 293 cells. Equal amount of cell lysates were loaded and separated by a 12% SDS gel under non-reducing condition. S-glutathionylated proteins were detected by Western blot using anti-glutathione monoclonal antibody. Some predominately modified proteins are indicated by arrows. (B) The same amount of lysates from 1(A) were separated in a SDS-gel under reducing condition (50 mM DTT) and the membrane was blotted and stained the same way as in 1(A). The loss of signals indicated the anti-glutathione monoclonal antibody was selectively detecting glutathione moiety on proteins.</p

The Effect of Diamide (0.5 mM) and Hydrogen Peroxide (0.5 mM) on Glutathione Concentration.

*<p>Total GSH is calculated by [GSH]+2[GSSG].</p