PRX3 turnover promotes adduction of specific cysteine residues by thiostrepton in cells.

(A) Reconstitution of the PRX3 catalytic cycle in vitro with purified components. (B) MM cells were treated with 5 μM TS for 18 hr or pre-incubated with 1 μM GV for 6 hr then treated with 5 μM TS (G/T) for 18 hr and immunoblotted for PRX3 after reducing SDS-PAGE. (C) MM cells were pre-incubated with 1 μM GV for the indicated times and then treated with 5 μM TS for 1 hr and cell lysates were immunoblotted for PRX3 after reducing SDS-PAGE. (D) Pre-incubation of MM cells with dimedone (Dim) for 6 hr blocked TS induced modification of PRX3. (E) HM Cells transfected with Flag-Tagged PRX3 expression plasmids were treated with 5 μM TS, lysates were collected at the indicated time points and TS induced modifications of PRX3 were visualized by immunoblotting with anti-PRX3 antibody after separation by reducing SDS-PAGE. See also S1 Fig.