FIG. 6.

Oxidative stress increases CBS S-glutathionylation and activity in HEK293 cells. (a) HEK293 cells were preincubated±250 μM BioGEE for 1 h and subsequently exposed to 200 μM H₂O₂ for 10, 30, and 60 min. Biotin-bound proteins were extracted using streptavidin affinity resin and eluted with 10 mM DTT. The eluents (top) and whole cell extracts (10 μg protein/lane) were subjected to nonreducing SDS-PAGE and probed with anti-CBS antibody. (b) Cell lysates from HEK293 cells exposed to 200 μM H₂O₂ for 10, 30, and 60 min were used to assay for CBS activity using methylcysteine in the presence of the CSE inhibitor, 2.5 mM PPG. CH₃SH production was measured by gas chromatography coupled to a sulfur chemiluminescence detector as described under the “Materials and Methods” section. The data are representative of at least three independent experiments. *p<0.05 or **p<0.01 versus control. BioGEE, biotinylated glutathione ethyl ester; CSE, cystathionine-γ-lyase; PPG, propargylglycine.