FIG. 6.
Reduction of in vitro-oxidized c-CRD by Trx2p. (A) Purified and reduced ΔGFP-CRD was treated with 0.2 mM (lanes 3 and 4) or 1.0 mM (lanes 5 and 6) H2O2 and was mixed without (lanes 3 and 5) or with (lanes 4 and 6) reduced Trx2p. SDS–15% PAGE was performed after the reaction with AMS (lanes 2 to 7 and 9) or was not performed (lanes 1, 8, and 10). Controls for the fully reduced ΔGFP-CRD (lanes 2 and 7) and Trx2 (lane 9) were performed by AMS treatment of reduced ΔGFP-CRD and Trx2, respectively, and the samples without AMS treatment were separated as the controls for fully oxidized ΔGFP-CRD (lanes 1 and 8) and thioredoxin (lane 10). (B) The ΔGFP-CRD oxidized with 2 mM diamide (lanes 3 and 4) was reduced by reduced thioredoxin (lane 4). The control for the reduced ΔGFP-CRD (lane 2), the oxidized ΔGFP-CRD (lane 1), reduced thioredoxin (lane 5), and oxidized thioredoxin (lane 6) was created as described above. Arrows indicate the positions of the reduced ΔGFP-CRD (CRDred), oxidized ΔGFP-CRD (c-CRDox), reduced thioredoxin (Trxred), and oxidized Trx (Trxox).