Quantifying PPAR-γ Expression in MSCs

Western blotting was performed in the cells as described previously [43 (link)]. The protein concentration was verified by the method of Lowry [44 (link)]. MSCs and MSCs-ω3 cells were lysed with RIPA lysis buffer (200 μL) per plate (100 mm²). For 5 min at 4 °C, the lysates of cells were centrifuged at 12,000 g, and the supernatants were stored at −80 °C. 30 μg of proteins were separated by 10% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad). The nonspecific binding sites were blocked in a TBS buffer with 5% albumin. The immunoblots were incubated overnight at 4 °C with GAPDH (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) and peroxisome-proliferator activator receptor gamma: PPAR-γ (1:500, Abcam, Cambridge, UK). After washing with TBS-T, the membranes were incubated for 1 h at 4 °C in HRP-conjugated secondary antibodies (1:30,000; Cell Signalling). Using Pierce ECL Plus Chemiluminescent substrate-detecting reagents (Thermo Fisher, Waltham, MA, USA), the immunoreactive protein bands were visualized. Images were obtained and analyzed with an Alliance 7 Chemiluminescence documentation system (UVItec, Cambridge, UK). The immunoblot band intensities were quantified using Image J software and expressed as the PPAR-γ/GAPDH ratio.