Af6284

After 14 days of osteogenesis or adipogenesis, the ADSCs were lysed with the RIPA cell lysate and protease inhibitor to extract the total protein. SDS-PAGE (Solarbio, China) was used to separate proteins with various molecular weights, and then, the separated proteins were blotted onto a PVDF membrane (Millipore, USA) using a transBlot turbo transfer system. After being sealed in 5% skimmed milk for 2 h, the membrane was incubated in the primary antibody overnight at 4 °C. The primary antibodies used are as follows: anti-COL1A1 AF7001, Affinity, USA, 1:1000), anti-ALP (DF6225, Affinity, USA, 1:1000), anti-OPN (AF0227, Affinity, USA, 1:2000), anti-BMP2 (AF5163, Affinity, USA, 1:1000), anti-RUNX2 (AF5185, Affinity, USA, 1:2000), anti-β-actin (AF7018, Affinity, USA, 1:10,000), anti-p-SMAD1/5/8 (AF8313, Affinity, USA, 1:2000), and anti-PPARγ (AF6284, Affinity, USA, 1:2000). Then, the membrane was incubated with the HRP-conjugated second antibody for 2 h and detected by ECL and super signal detection agents (Thermo Fisher Scientific, USA).

The homogenate of liver tissue or cells was prepared in lysis buffer (20 mM Tris–Cl pH7.5, 140 mM NaCl, 1 mM CaCl₂ and MgCl₂, 10 mM NaF, 1% NP-40, 10% glycerol, 2 mM Na-Vanadate, and 1 mM PMSF) supplemented with complete Protease Inhibitor Cocktail (cOmplete™, Sigma-Aldrich, Dallas, TX) for 30 min, centrifuged at 12,000 rpm at 4°C for 15 min. The protein samples were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membranes. The membranes were blocked at room temperature for 2 h in 5% defatted milk dissolved in TBST (10 mM Tris–HCl, pH 7.4, and 150 mM NaCl), incubated at 4°C for overnight with the following antibodies: PPARγ (#AF6284, Affinity, 1:1000), FGF9 (#A6374, ABclonal, 1:500 and #ab206408, Abcam, 1:500), ChREBP (#A7630, ABclonal, 1:1000), Fasn (#DF6106, Affinity, 1:1000), or β-Actin (#AF7018, 1:10000), washed in TBST (0.1%Tween 20) for 15 min (repeated three times) and incubated with a goat anti-mouse (# S0002, Affinity) or goat anti-rabbit (# S0001, Affinity) IgG (H + L) HRP secondary antibody (1:5000 dilution in TBS) for 1 h at RT. Immunoreactivity was visualized and quantified by infrared scanning using the Odyssey system (LI-COR Biosciences).