*2.12. Western Blot*

After washing the cells with PBS, they were lysed with RIPA buffer supplemented with protease inhibitor cocktail (Thermo Fisher Scientific). The Bradford assay was used to estimate the total protein concentration. Proteins (60 μg) were electrophoresed in 10% or 12% SDS-polyacrylamide gel and then transferred to PVDF membrane (EMS–Millipore, Billerica, MA, USA). The blots were then blocked with 3% skim milk or BSA in Tris-buffered saline (TBS)-Tween 20 for 1 h, incubated overnight with protein-specific primary antibodies [TTR (1:400), MYOD (1:500), MYOG (1:500), D2 (iodothyronine deiodinase type 2; 1:500), RXRγ (1:500) (Santa Cruz Biotechnology) or β-actin (1:2000) antibody (Santa Cruz Biotechnology), TR-α (1:500, Thermo Fisher Scientific), MYL2 (myosin light chain 2, 1:1000, Abcam, Cambridge, MA, USA) or FNDC5 (1:500, Bioss Antibodies, Woburn, MA, USA)] in 1% skim milk or BSA in TBS at 4 ◦C. The blots were then washed and incubated with horse radish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature and then developed with Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). Supplementary Table S3 shows the molecular weight of protein.
