Fig. 6.

Identification of RTS^GS targets by the BTA assay in vitro. A, Schematic of workflow to identify cysteine residues within the proteome, candidates for regulation by the RTS^GS. Cell lysates were pretreated with TCEP to remove any reversible cysteine-based protein modifications, then incubated with GSSG to incorporate S-glutathionylation and disulfides to proteomes. The GSSG-treated protein extracts were treated with N-ethylmaleimide (NEM) to block any free thiol groups. After desalting, the lysates were exposed to H₂S (NaHS) followed by the BTA assay. The RTS targets were analyzed in a SDS gel or by LC-MS. B, The BTA assay was performed to detect RTS targets in extracts from mouse liver. GSSG-treated mouse liver lysates were divided into two equal fractions, then treated without or with an H₂S donor (NaHS, 2 mm) for 1h followed by the BTA assay. The eluates were analyzed by SDS-PAGE electrophoresis and silver staining of proteins. C, Mouse liver lysates (equal amount/treatment) were treated and analyzed as in B but with increasing concentrations of the H₂S donor-NaHS (10, 20, 40, 80, 100, and 200 μm).