Figure 5.

The Srx–TXNDC5 interaction is not affected by the treatment of cells with exogenous H₂O₂. A, HEK293-FLAGSrx or A549 cells were treated with vehicle or increasing concentrations of H₂O₂ for 10 min. Cell lysates were collected, and IPs were performed using anti-FLAG or anti-Srx antibodies. IP eluates were separated by SDS-PAGE under reducing conditions. Western blotting results indicate that treatment of cells with H₂O₂ does not affect the amount of endogenous TXNDC5 pulldown by FLAG-Srx in HEK293T or Srx in A549 cells. B, IP eluates from A were separated by SDS-PAGE under nonreducing conditions, and Western blotting indicates the position of monomer as well as possible disulfide bond formation between Srx and TXNDC5 as bands indicated by an asterisk. C, FLAG-Srx or its cysteine mutant (C99A) was expressed in HEK293T cells. Cell lysates were collected and IPs were performed using anti-FLAG antibody. IP eluates were separated by SDS-PAGE under reducing conditions. Western blotting results indicate that mutation of cysteine 99 in Srx does not affect its ability to interact with TXNDC5.