Western blot detection system

D3G (chemical formula C₂₁H₂₁O₁₂Cl, >97% purity) was purchased from Polyphenols AS (Sandnes, Norway). Dorsomorphin, AICAR, indomethacin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone (DEX) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), bovine calf serum (BCS), trypsin-EDTA, and fetal bovine serum (FBS) were procured from Thermo Fisher (San Jose, CA, USA). CCK-8 was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Insulin was purchased from Thermo Fisher (San Jose, CA, USA). Random hexamers and the reverse transcriptase enzyme mix (GoScriptTM) were obtained from Promega (Madison, WI, USA). Monoclonal antibodies against C/EBPα, SREBP1, PPARγ, FAS, ACC, SIRT1, p-ACC, AMPK, p-AMPK, CPT-1, and horseradish peroxidase (HRP)-coupled anti-rabbit or anti-mouse secondary antibodies were purchased from Abcam (Cambridge, UK). Total RNA was extracted using an easy-spin^TM Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam-si, Korea) and a PRO-MEASURE^TM protein measurement solution, protein lysis buffer, and a Western blot detection system were purchased from iNtRON Biotechnology (Seongnam-si, Korea). The polyvinylidene difluoride (PVDF) membrane was procured from Merck (Burlington, Massachusetts, USA).

Cells were treated with the specified concentrations of CTS for 24 h, harvested, washed with PBS, and recentrifuged (1,890 × g, 5 min, 4°C). The resulting cell pellets were resuspended in lysis buffer containing 50 mM Tris (pH 7.4), 1.5 M sodium chloride, 1 mM EDTA, 1% NP-40, 0.25% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and a protease inhibitor cocktail. The cell lysates were incubated on ice for 1 h and clarified by centrifugation at 17,010 × g for 30 min at 4°C. Protein content was quantified using a Bradford assay (Bio-Rad, Hercules, CA, USA) and a UV spectrophotometer. The cell lysates were separated by 10–12% SDS polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene difluoride membranes (PVDF; Millipore, Billerica, MA, USA), which were blocked in 5% non-fat dried milk dissolved in Tris buffered saline containing Tween-20 (2.7 M NaCl, 53.65 mM KCl, 1 M Tris-HCl, pH 7.4, 0.1% Tween-20) for 1 h at room temperature. The membranes were incubated overnight at 4°C with specific primary antibodies. After washing, the membranes were incubated with the secondary antibodies (HRP conjugated anti-rabbit or anti-mouse IgG) for 1 h at room temperature. After washing, the blots were analyzed using West-Zol Plus and a western blot detection system (iNtRON Biotechnology, SungNam, South Korea).