R4100

The 3T3-L1 cells induced to differentiate were suspended in radioimmunoprecipitation assay buffer (RIPA; R4100, GenDepot, USA) at 4°C for 30 min, and the resulting solution was centrifuged at 14,000 rpm for 30 min to obtain cell lysates.
Cell lysates were analyzed using a protein assay kit (BR500-022, Bio-Rad, USA) to measure protein contents, and these samples were further analyzed using 10% SDS-PAGE (BR456-8033, Bio-Rad, USA). After separation on SDS-PAGE, the proteins were transferred to a PVDF membrane (BR170-4156, Bio-Rad, USA), which was blocked with 5% skim milk for 1 h. Primary anti-PPARG (1:1000), anti-CEBPA (1:500), anti-SREBF1(1:1000), and anti-GAPDH (1:2500) antibodies were added, and the samples were incubated for 15 h at 4°C. Subsequently, the membranes were hybridized with the secondary horseradish peroxidase-conjugated anti-rabbit IgG (SA002-500) or anti-mouse IgG (SA001-500, GenDepot) antibodies for 1 h at 25°C. Finally, the membranes were incubated with the chemiluminescent substrates, and the protein bands were imaged using the ChemiDoc MP imaging system (Bio-Rad, USA).

Cells were lysed in radioimmunoprecipitation assay buffer (R4100; GenDEPOT, Katy, TX, USA) containing protease and phosphatase inhibitors (Sigma-Aldrich). The soluble fraction of cell lysates was isolated by centrifugation at 13,000 rpm for 30 min at 4 °C. The protein concentration was measured using a protein assay (Bio-Rad, Hercules, CA, USA) and 20–40 μg was resuspended in Laemmli buffer and incubated for 5 min at 95 °C before proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). After incubation in Tris-buffered saline containing 0.1% Tween 20 and 5% bovine serum albumin (BSA) for 1 h, the blot was incubated overnight at 4 °C with primary antibody. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody for 1 h, and protein bands were visualized by enhanced chemiluminescence (ElpisBiotech, Daejeon, Korea). Uncropped images of blots are shown in Supplementary Fig. 15.