Fig. 4.

2DE analysis of biotinylated proteins from vector (Vec)- (A) and Trx1^C32S/C35S (B)-transfected HeLa cells is shown. Nitrosylated proteins were biotinylated, enriched and eluted from streptavidin beads, desalted with ice-cold acetone, dissolved in a 2DE rehydration buffer, and analyzed by 2DE. Gels were stained with SYPRO Ruby dyes, and the protein spots whose densities were increased in Trx1^C32S/C35S-transfected cells were excised for trypsin digestion and protein identification by tandem MS (supplemental Table SI). C, proteins recovered from streptavidin beads were analyzed by Western blotting with individual antibodies specific for the detection of nitrosylated Trx1, Prx1, HSP90β, actin, Casp3, Txnip, and TrxR. Values are the mean ± S.E. for experiments performed in triplicate (*, p = 0.0006; †, p = 0.001; ‡, p = 0.0001; §, p = 0.0007; , p = 0.001; #, p = 0.0009; **, p = 0.002; Student's t test). Non-streptavidin-enriched proteins were analyzed in parallel to detect proteins regardless of their nitrosylation status.