Sc 6529

Mechanically homogenized tissue samples were boiled for 10 min in gel-loading buffer (125 mM Tris-HCl, 4% sodium dodecyl sulfate [SDS], 10% 2-mercaptoethanol, pH 6.8, 0.2% bromophenol blue). Total protein equivalents for each sample were separated by SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes. Immunoblotting was performed with antibodies against SMP30 (a kind gift from Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology), UCP1 (SC-6529; Santa Cruz Biotechnology, Dallas, TX, USA), β-actin (SC-47778; Santa Cruz), GAPDH (SC-32233; Santa Cruz), TFIIB (SC-271736; Santa Cruz), PPAR-α (ab24509; Abcam), and FGF21 (AF3057; R&D Systems, Minneapolis, MN, USA or SC 292879). Western blots were visualized by enhanced chemiluminescence (Davinch-Chemi System, CAS400) according to the manufacturer’s instructions. The SMP30 antibody was validated using liver samples from WT and SMP30-KO mice.

Protein samples were extracted from tissue homogenized in Radio Immunoprecipitation Assay (RIPA) buffer with Protease Inhibitor Cocktail (Dingguo, China). Aliquots containing 10 μg of proteins were loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel, transblotted onto a polyvinylidene difluoride membrane (Bio-Rad), blocked with 5% BSA in Tris-buffered saline with 0.1% Tween-20, and then incubated with the primary antibodies for UCP1 (1:1000, Santa Cruz, sc-6529), peroxisome proliferator activated receptor gamma (PPARγ) (1:1000, Santa Cruz, sc-6285), PPARγ coactivator 1-alpha (PGC-1α) (1:1000, Santa Cruz, sc-13,067), and PR domain containing 16 (PRDM16) (1:1000, Absin, abs104818), PKA (1:1000, cell signaling technology, 4782S), p-PKA (pThr197) (1:1000, cell signaling technology, 4781S) and β-ACTIN (1:2000, Santa Cruz, sc-47,778). Protein visualization was used and the electrochemiluminescence (ECL) detection reagents and films were exposed on the chemiluminescence imaging system (Tanon, China) analyzed using the Image-J. Relative protein expression was normalized with the expression level of β-ACTIN.

After sWAT sample deparaffinization, antigen retrieval and nonspecific binding blockade were performed as previously described [12 (link)]. Sections were incubated with anti-UCP1 (anti-goat, SC-6529, Santa Cruz Biotechnology) primary antibody and secondary anti-goat Alexa Fluor 546 conjugated antibodies were used. Slides were mounted with Slow Fade (Invitrogen, Molecular Probes, Carlsbad, CA, USA) to maintain fluorescence. Control sections were obtained after the omission of the primary antibodies. A fluorescence microscope (BX51, Olympus America Inc., Florida, USA) with a DP71 camera was used to assess the slides.