Ab174302

The CBS/CSE inhibitor AOAA (O-(carboxymethyl)hydroxylamine hemihydrochloride) [9 (link)] was purchased from Sigma-Aldrich (Burlington, MA, United States), the 3-MST inhibitor HMPSNE (2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one) [10 (link)] was purchased from MolPort (Riga, Latvia). The fluorescent H₂S probe 7-azido-4-methylcoumarin (AzMC) was obtained from Sigma-Aldrich (Burlington, MA, USA).
Rabbit monoclonal anti-CBS (D8F2P), anti-ATP citrate lyase (ACLY) (D1 × 6P), anti-β-catenin (D10A8), anti-mouse IgG HRP-linked antibody (#7076) and rabbit polyclonal anti-phospho-ACLY (Ser455) were purchased from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal anti-β-actin (AC-15) and rabbit polyclonal anti-SQR (HPA017079) were obtained from Sigma-Aldrich (Saint Louis, MO, USA). Anti-rabbit IgG (H+L) cross-adsorbed secondary antibody-HRP (#31458) was purchased from Invitrogen (Thermo Fisher Scientific, Waltham, MA, USA). Rabbit polyclonal anti-3-MST (ab224043), anti-CSE (ab151769), anti-TST (ab231248) and rabbit monoclonal anti-ETHE1 (ab174302) were purchased from Abcam (Cambridge, UK).

Pre-adipocytes were seeded at a density of 6 × 10⁴ cells per well in 6-well plates. Following differentiation and treatments, cells were washed twice with PBS and incubated on ice with lysis buffer (ELISA lysis buffer, Thermo Fisher Scientific) containing proteinase and phosphatase inhibitor (Cell Signaling technology, Leiden, The Netherlands). Proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane (Thermo Fisher Scientific), and incubated with specific primary antibodies. The antibodies used in this study were directed against β-actin (Cell Signaling Technology, 8H10D10, 1:2,000), CBS (Cell Signaling Technology, D8F2P, 1:500), CSE (Abcam, Ab151769, 1:1,000), 3-MST (Abcam, Ab85377, 1:500), ETHE-1 (Abcam, Ab174302, 1:1,000), TST/rhodanese (Abcam, Ab231248, 1:1,000), and PPAR-γ (CST, 2430S, 1:1,000). Signals from HRP-coupled secondary antibodies were detected with ECL Prime Western Blotting Detection Reagent (Sigma-Aldrich), using a luminescent image analyzer (Fusion FX6, Vilber Lourmat, Marne la Vallée, France) and quantified using the ImageJ software (NIH, Bethesda, MD, USA).