The signals of protein bands were reduction sensitive, in the presence of DTT, indicating that this monoclonal antibody selectively detected protein mixed-disulfided with glutathione. The overall pattern of S-glutathionylation suggests different degrees of sensitivities of proteins in response to oxidative stress. The predominately S-glutathionylated proteins may be due to the number of reactive cysteine residues associated with protein, or simply, the reflection of the abundance of a protein level. Based on the presented data, our results suggest that different oxidative mechanisms stimulate different sets of proteins Usaramine responding to stress. In our studies, even in the presence of more reduced GSH than GSSG. Our data supports a previously proposed Cyclosporine mechanism where glutathione disulfide is not a prerequisite factor for the formation of S-glutathionylation in cells. In theory, diamide should induce all possible proteins. However, a significantly modified protein is only induced by hydrogen peroxide. These data suggest the 43 kDa protein is only sensitive to a free radical related mechanism because another oxidant, t-Butyl hydroperoxide in HEK 293 cells. To our knowledge, this is the first evidence that shows specific protein S-glutathionylation by different oxidative mechanisms in cells. It is necessary to determine the identity of protein to understand its function and propose a mechanism for the specific by hydrogen peroxide. We speculate that this protein could be actin since actin has been shown as a target protein. After oxidant treatment, the cells were harvested in 5% perchloric acid for 20 min on ice. Acid soluble and insoluble fractions were separated by centrifugation. Acid soluble fractions were used to determine GSH and GSSG concentration and acid insoluble fractions were used to determine the protein concentration. Glutathione samples were prepared and determined by a previously described method. Briefly, acid-soluble fractions were reacted with iodoacetic acid at neutralized pH to block free sulfhydryal groups. Thus, any additional drug resistance generated by these individuals is likely to make only a small contribution to the incidence rate.