Ab85377

Polyclonal antibodies to 3-MST (ab85377) and CSE (ab151769) were from Abcam and used at a concentration of 1:1,000. Anti-rabbit secondary antibody (7074) was used at a concentration of 1:10,000 in TBS-T and was from Cell Signaling Technology.

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).