Fig. 4.

Cysteine-32 in Trx1 is responsible for Trx1 S-desulfhydrating proteins. A and B, cysteine-32 mutation abolished the inhibitory role of Trx1 on protein S-desulfhydration. HEK293 cells were transfected with Trx1 cDNA and mutated Trx1 cDNA (C32S and C35S) for 24 h following incubation with 50 µM NaHS for 2 h. After that, the cells were collected for biotin switch assay using antibody against PC (A) and GAPDH (B). *<0.05 versus the control without NaHS treatment. n=4. C and D, cysteine-32 mutation disassociated Trx1 binding with S-sulfhydrated PC and GAPDH. HEK293 cells were transfected with Trx1 cDNA and mutated Trx1 cDNA (C32S and C35S) for 24 h following incubation with 50 µM NaHS for 2 h. After that, the cells were collected for co-immunoprecipitation assay as indicated in the figure. n=3.