β had

The expression of proteins directly involved in lipogenesis (Fas; Cell Signaling, Beverly, MA, USA), oxidation pathway (Cpt1, β-had; Santa Cruz Biotechnology, Dallas, TX, USA) and the process of desaturation and elongation (Elovl3, Elovl6, Scd1; Santa Cruz Biotechnology) as well as fatty acid exporting proteins: Abca1 (Thermo Scientific, Waltham, MA, USA) and Mtp (Santa Cruz Biotechnology) were detected by routine Western Blotting as previously described in details by Konstantynowicz-Nowicka et al. [28 (link)]. Briefly, protein concentration was determined using bicinchonic acid method (BCA) with bovine serum albumin (BSA) as a standard. Signals obtained by immunoblotting were quantified densitometrically using a ChemiDoc visualization system (Bio Rad, Warsaw, Poland). Equal protein loading was confirmed using Ponceau S staining. The expression of all the proteins was standardized to the Gapdh (Santa Cruz Biotechnology) expression and the control was set at 100%.

The ADMSCs-derived adipocytes were lysed in RIPA buffer containing protease and phosphatase inhibitors (Roche Diagnostics GmbH, Mannheim, Germany). The protein content in the lysates was measured using the BCA method with bovine serum albumin (fatty acid–free, Sigma-Aldrich, St. Louis, MO, United States) as the standard. Before protein separation using SDS-PAGE (the same amounts of protein, i.e., 30 μg was loaded on Criterion TGX Stain-Free Precast Gels, Bio-Rad, Hercules, CA, United States) and being transferred to the PVDF membrane, the samples were denatured in Laemmli buffer (Bio-Rad, Hercules, CA, United States). Finally, the membranes with the transferred proteins were blocked in 5% non-fat dry milk in Tris-buffered saline–Tween and incubated with the corresponding primary antibodies: AS160 (Merck Millipore, CA, United States), DGAT1, β-HAD, ATGL, GAPDH (Santa Cruz Biotechnology, Inc., Dallas, TX, United States), and FASN (Abcam, Cambridge, UK). Binding of the primary antibody was detected after incubation by using HRP-conjugated secondary antibody and then Clarity or Clarity Max Western ECL substrates (Bio-Rad, Hercules, CA, United States). Protein bands were quantified densitometrically by using the ChemiDoc Imaging System (Bio-Rad, Hercules, CA, United States), and their expression was normalized to GAPDH reference protein expression.