Fig 2.

TpxD cysteine-thiol redox state under aerobic conditions. D39 cells were resuspended in AMS buffer supplemented with 20 mM AMS, sonicated, and incubated at room temperature for 2 h. Alkylated samples were loaded onto 18% (wt/vol) polyacrylamide–SDS gels and immunoblotted by using TpxD polyclonal antibodies. Reduced samples were prepared by incubating bacterial lysates with 50 mM DTT and then alkylated as described above. Lane 1, unalkylated lysates (no AMS added); lane 2, AMS-treated bacterial lysates; lane 3, lysates treated with DTT prior to AMS alkylation. Experiments were repeated 3 times.