Western Blot Analysis of Thermogenic Proteins

Proteins in SAT were extracted by lysing in modified radioimmunoprecipitation buffer (Thermo Fisher, Waltham, MA, USA), as previously described [19 (link)]. An equal amount of protein was loaded and separated using electrophoresis gels (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinylidene fluoride membranes. Blocker™ FL Blocking Buffer (Thermo Fisher, Waltham, MA, USA) was used to block the membrane along with primary antibodies for TBP (1:1000, Cell Signaling, Danvers, MA, USA) as a housekeeping control and UCP1 (1:1000, Thermo Fisher Scientific, Rockford, IL, USA), CIDEA (1:1000, Abcam, Cambridge, MA, USA) and FGF21 (1:1000, Abcam, Cambridge, MA, USA) as target genes. Rabbit polyclonal antibody was used as a secondary antibody (1:25,000). Blots were developed using Li-COR Imager System (Lincoln, NE, USA).