Figure 7.

Sensitivity and response to ROS of WT and STXA2 strains. A, Time course of relative ROS amount in the WT and STXA2 before (0 h) and after inducer removal (24–72 h) using the CM-H₂DCFDA reagent. B, In vivo redox state of 2-Cys Prx in the WT and STXA2 strains. Cells were collected before (0 h) and after 48 h of inducer removal (48 h). The pellets were resuspended in 1× Laemmli buffer and boiled for 10 min, then 20 μL of the boiled cell suspension were loaded on a SDS–PAGE gel under nonreducing conditions, except for a sample of the WT strain that was also treated with DTT 10 mM as a control. 2-Cys Prx was detected by Western blot using specific antibody. C, Reduction level of 2-Cys Prx is defined as the ratio of the reduced form (monomer) and the sum of reduced and oxidized forms (monomer and dimer respectively). D, Western blot analysis of GrxA, GrxC, and 2-Cys Prx before (0 h) and after inducer removal (24–72 h). Samples equivalents to 4 ×10⁷ cells were taken at the indicated times and total proteins were isolated and resolved on SDS–PAGE, blotted and incubated with specific antibodies. E, Quantification of the indicated proteins in the STXA2 strain before inducer removal (0 h) relative to the WT (dashed line). F, Quantification of the indicated proteins in the STXA2 strain after 48 h inducer removal relative to the signal with inducer (0 h; dashed line). Data are means ± sd from three biological replicates in all cases. **P < 0.01 (two-tailed Student’s t test between 0 and 48 h).