Ab8934

After decapitation, the right portion of interscapular BAT was frozen immediately. Later, the protein content was estimated by the method of Lowry et al. [18 (link)]. Primary antibodies against Pex11β (1:1000, ab74507), Pex19 (1:2000, ab137072), Pex16 (1:500, sc-398189), Pex5 (1:1000, sc-137103), Pex13 (1:1000, sc-271477), Pex26 (1:500, sc-376817), Pex6 (1:500, sc-271813), PMP70 (1:1000, ab74507), catalase (1:800, ab1877), calnexin (1:1000, ab22595), dynamin-related protein 1—Drp1 (1:1000, ab93942), PPARα (1:2000, ab8934), and PPARγ (1:400, ab19481) were purchased from Abcam (Abcam, Cambridge, UK) or Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA). All used antibodies were chosen as they recognize the exact epitope, a small amino acid sequence, which increases their specificity toward the target protein. Immunoreactive bands were quantified using ImageJ software (NIH, Bethesda, MD, USA). The volume represents the sum of all pixel intensities within a band, and 1 pixel = 0.007744 mm². We averaged the ratio of pixels per band for the target protein in the corresponding samples from three similar independent experiments. The mean values obtained from the euthyroid group were taken as 100%, and those from methimazole-treated groups were expressed as percentages against the euthyroid group.

Tissues were fixed with 10% (v/v) neutral buffered formalin. One part of each liver was embedded in a paraffin block, and the other part was frozen with Tissue-Tek O.C.T. Compound (Sakura) after cryoprotection with 30% sucrose. Paraffin-embedded livers were sliced into 5-μm sections and stained with hematoxylin and eosin (H&E) for immunostaining in this study. Frozen liver sections were cut into 5-μm sections and stained with Oil red O (Sigma) in 60% isopropanol or 0.25 mg/ml filipin. Paraffin-embedded inguinal white adipose tissue was sliced into 5-μm sections and stained with H&E or CellMask Orange Plasma membrane Stain (Thermo Fisher) with DAPI for nuclear staining. Adipocyte area and adipocyte counting were measured using ImageJ software in 6–8 different fields and are presented in a bar graph. For immunostaining, sections were incubated with the following primary antibodies: ACOT12 (MBS273137, Mybiosource), FASN (GTX109833, GeneTex), SCD1 (ab19862, Abcam), PPARα (ab8934, Abcam), and HA-tag (3724, Cell Signaling). Positive staining was visualized by detection with DAB Peroxidase (HRP) Substrate (SK-4105, Vector Laboratories), and counterstaining with hematoxylin. Histological images were acquired with a light microscope (Thermo Fisher).

Whole cell protein was extracted from frozen liver tissue, and total protein lysates were subjected to SDS-PAGE on 8–20% acrylamide gels, electro-transferred to polyvinylidene difluoride membranes (MilliporeSigma, Burlington, MA, United States), and probed overnight at 4°C in the presence of the following primary antibodies: anti-SIRT1 (1:1,000, 13161-1-ap, Proteintech Group, Inc., Rosemont, IL, United States), anti-PPARα (1:1,000, ab8934, Abcam, Cambridge, United Kingdom), and anti-SREBP (1:1,000, bs-1402R, Bioss Antibodies, Woburn, MA, United States). For protein detection, we used horseradish peroxidase-conjugated secondary antibodies (Wuhan ServiceBio Technology Co., Wuhan, China). Five samples per group per timepoint were assayed; protein levels were normalized to β-actin (1:3,000, GB12001, Wuhan ServiceBio Technology Co.). Densitometry analysis of protein bands was conducted using the open-source image processing software ImageJ (https://imagej.net/ImageJ).

Liver samples were lysed in ice-cold RIPA buffer supplemented with 1 mM PMSF (Sigma, St. Louis, MO, USA) and then kept at 4 °C for 2 h. Supernatant was collected by centrifuge at 10,000× g for 15 min at 4 °C. For SDS–PAGE, samples were mixed with SDS sample buffer and incubated at 98 °C for 5 min. Western blot was performed with the following antibodies: β-actin mAb (#3700, Cell Signaling, Danvers, MA, USA), peroxisome proliferator activated receptor α (PPARα) pAb (ab8934, Abcam, Cambridge, UK), sterol regulatory element-binding transcription factor 1 (SREBP1) mAb (sc-365513, Santa Cruz Technology, Santa Cruz, CA, USA), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) mAb (ab174830, Abcam).