The assay was carried out as described previously (8 (link)), with modifications. Briefly, liver tissues or cells were homogenized in HEN buffer [250 mM Hepes-NaOH (pH 7.7), 1 mM EDTA, and 0.1 mM neocuproine] supplemented with 100 μM deferoxamine and centrifuged at 13,000g for 30 min at 4°C. Cell lysates (240 μg) or pure GAPDH protein (0.3 μg), treated with CSE, L-cysteine, or NaHS where indicated, were added to blocking buffer (HEN buffer adjusted to 2.5% SDS and 20 mM MMTS) at 50°C for 20 min with frequent vortexing. The MMTS was then removed by acetone and the proteins were precipitated at −20°C for 20 min. After acetone removal, the proteins were resuspended in HENS buffer (HEN buffer adjusted to 1% SDS). To the suspension was added 4 mM biotin-HPDP in dimethyl sulfoxide without ascorbic acid. After incubation for 3 hours at 25°C, biotinylated proteins were precipitated by streptavidin-agarose beads, which were then washed with HENS buffer. The biotinylated proteins were eluted by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and subjected to Western blot analysis. For quantitation of protein sulfhydration, samples were run on blots alongside total lysates (“loads”) and subjected to immunoblotting with antibodies specific to each protein. The sample to load ratio was then densitometrically analyzed with the software programs EagleSight 3.2 (Stratagene) and Odyssey 2.1 (Li-Cor).