RTCB is a substrate of TRX. (A) Scheme of the reduction mechanism of TRX CxxC toward its target protein containing an oxidized cysteine residue (left panel). Trapping of target proteins using TRX CxxS (middle panel). The double mutant SxxS cannot reduce the oxidized cysteine on the target protein due to mutation of the catalytic cysteines (right panel). (B) shTRX cells constitutively overexpressing FLAG-TRX CxxS were treated for 3 min with increasing concentrations of H2O2 before lysis. ShTRX cells were left untreated as a negative control. Immunoprecipitation and elution were performed in nonreducing conditions. Eluates were analyzed by western blot using anti-RTCB, anti-PRDX1, and anti-TRX antibodies. (C) Trapping was performed as described in B after treatment with increasing concentrations of menadione for 15 min. ShTRX cells were left untreated as a negative control. (D)Trapping was performed as described in B in shTRX cells expressing FLAG-TRX CxxS after treatment with 100 µM H2O2 for 3 min or 40 µM menadione for 15 min. Untreated shTRX and shTRX overexpressing FLAG-TRX CxxS were used as controls. Inputs and eluates were analyzed by western blot using anti-DDX1, anti-RTCB, anti-FAM98B, anti-CGI-99, anti-PRDX1, and anti-TRX antibodies. (E) Trapping was performed as described in B in shTRX cells expressing FLAG-TRX CxxC, FLAG-TRX CxxS, and FLAG-TRX SxxS after treatment of cells with 0 or 100 µM H2O2 for 3 min.
