2.9. Biotin switch assay for protein sulfhydration

The assay was performed as described by Mustafa et al. 14 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. Homogenates were sonicated and centrifuged at 12,000 rpm (4°C) for 10‐15 min, treated with DTT, NaHS or GYY4137 for in vitro sulfhydration. After cell treatment with DTT, NaHS or GYY4137, or transfection with adenovirus, cell homogenates were collected for in vivo sulfhydration. Cell lysates were added blocking buffer (HEN buffer adjusted to 2.5% SDS and 20 mM MMTS) then incubated at 50℃ for 20 min with frequent vortex. The MMTS was then removed by acetone, and the proteins were precipitated at −20℃ for 20 min. Resuspended proteins in HENS buffer (HEN buffer adjusted to 1% SDS) and added in 4 mM biotin‐HPDP for 3 hours at 25℃, incubated overnight with magnetic beads. Then, biotinylated protein was pulled down by streptavidin magnet beads and eluted by SDS‐PAGE loading buffer for Western blot analysis.