FIGURE 4.

Thioredoxin-interacting protein binds to the active site Cys of Trx1 and facilitates protein S-nitrosylation. A, HEK293 cells were transfected with pEBG-GST-Trx1(WT) and C32S active site mutant for 20 h, Trx1 was isolated with GSH-agarose and eluted with 50 mm dithiothreitol to release disulfide-linked Txnip. B, HEK293 cells stably transfected with either pcDNA3.1 or pcDNA3.1-Txnip were co-transfected with pLP or pLP-iNOS for 20 h. Protein-SNO was measured by mercury-coupled photolysis-chemiluminescence. **, p < 0.01. C, protein-SNOs were assayed by the BST following co-transfections as in B. Total protein S-nitrosylation (biotinylation) was detected with avidin-horseradish peroxidase. Indicated is a single representative experiment with in-gel cropping. D, HEK293 cells stably transfected with pCDNA3.1-Txnip were co-transfected ± iNOS and either empty pEBG or pEBG-GST-Trx1 and subjected to the BST as in C. E, S-nitrosylation of GAPDH was detected by the BST using Western blotting for GAPDH. IP, immunoprecipitation; IB, immunoblot.