Pparγ

Cells were seeded in 60‐mm plastic dishes (Costar) for total protein isolation. Proteins were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to a polyvinylidene fluoride membrane using a semidry transfer apparatus (Hoefer) for 1.5 hours at room temperature. Membranes were blocked with 5% milk in Tris‐buffered saline mixed with tween20 (TBST) for 2 hours at 37°C, and incubated with primary antibodies against TAZ (1:1000, Cell Signalling, USA), RUNX2 (1:200, Boster, China), OCN (1:200, Boster, China), C/EBPβ (1:200, Boster, China), PPARγ (1:200, Boster, China), or GAPDH (1:3000, Bioworld, USA) at 4°C overnight. Then the membranes incubated with IRDye800^® conjugated secondary antibody (1:20,000, Rockland, USA) for 1 hour at 37°C, following scanning with the Odyssey Infrared Imaging System (Li‐COR Biosciences). Then the integrated intensity for each detected band was determined with Image J, v.1.46.

Total cellular protein was isolated from adipocytes with a protein extraction kit (Solarbio Company, Beijing, China) after mixing with protein loading buffer and denaturation for 10 min in a 100 °C metal bath. A 20-µg protein sample was electrophoresed on a 12% gel, and the protein was transferred to a PVDF membrane after gel electrophoresis. Next, the membrane was incubated with antibodies against β-ACTIN (1:5000, NOVUS, HK, NP_776404.2), TP53INP2 (1:2000, AVIVA Systems Biology, San Diego, CA, USA, XP_003586891.1), PPARγ (1:1000, Boster, Wuhan, China, NP_851367.1), PLIN2 (1:2000, Abcam, Cambridge, UK, NP_776405.1), FASN (1:2000, Abcam, NP_777087.1), p62 (1:2000, Abcam, NP_788814.1), or LC3 (1:2000, Abcam, NP_001001169.1) for 12 h at 4 °C. After washing the membrane three times (10 min each) with PBS-Tween 20, the membrane was incubated with the secondary antibody for 1 h and then washed three times with PBS-Tween 20 (10 min each). Finally, equal amounts of luminol reagent and peroxide solution were mixed in an EP tube and added dropwise to the PVDF membrane. The Gel Doc™ XR+ Gel Documentation System (Bio-Rad, Hercules, CA, USA) was used to detect the immunoreactivity.