Blocker fl blocking buffer

Proteins were extracted from livers by homogenizing in modified radio-immunoprecipitation (RIPA) assay buffer (Thermo Fisher Scientific Inc.). Protein was loaded in equal amounts per lane and separated using TGX Stain-Free™ FastCast™ Gels (Bio-Rad) of appropriate gradients and transferred to a polyvinylidene fluoride (PVDF) membrane using Immobilon-FL Transfer Membranes (MilliporeSigma, Burlington, MA, USA). The PVDF membrane was blocked using Blocker™ FL Blocking Buffer (Thermo Fisher Scientific Inc.) for an hour followed by incubation with primary antibodies for fatty acid synthase (FASN) (Santa Cruz Biotechnology, CA, USA; dilution 1:1000) and phosphorylated (Thr172) [27 (link)] and total AMP-activated protein kinase (AMPK) (Cell Signaling Technologies, Danvers, MA, USA; dilution 1:500). Protein concentrations were normalized to TATA-Box binding protein (TBP; Cell Signaling Technologies; dilution 1:1000). Mouse polyclonal antibody was used as a secondary antibody for FASN (dilution 1:25,000), and rabbit polyclonal antibody was used as a secondary antibody for AMPK (dilution 1:25,000).

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).