Ab77481

Proteins were separated by SDS–polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes, and probed with primary antibodies A-FABP (0.25 μg ml⁻¹, goat polyclonal; AF1443, R&D Systems), UCP-1 (0.5 μg ml⁻¹, rabbit polyclonal; ab10983, Abcam, Cambridge, UK), liver X receptor α (LXRα, 1 μg ml⁻¹, rabbit monoclonal; ab28478, Abcam), type II iodothyronine deiodinase (D2, 0.25 μg ml⁻¹, rabbit polyclonal; ab77481, Abcam), tyrosine hydroxylase (0.25 μg ml⁻¹, rabbit polyclonal; 2,792, Cell Signaling), β-tubulin (0.25 μg ml⁻¹, rabbit polyclonal; 2,128, Cell Signaling, Beverly, MA, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 0.1 μg ml⁻¹, rabbit monoclonal; 5,174, Cell Signaling). The intensities of protein bands were quantified using the NIH Image J software.
Total RNA was extracted with Trizol (Invitrogen) and reverse transcribed into complementary DNA using Improm-II reverse transcription kit (Promega, Madison, USA). Real-time PCR was performed using SYBR Green master mix (Qiagen, Venlo, the Netherlands) on a 7,900 HT (Applied Biosystems, CA, USA), normalized against the GAPDH gene. Primer sequences are listed in Supplementary Table 1.

Protein samples were isolated from tissue or cells with lysis buffer^{51 (link),65 (link)}. Homogenized protein lysates were quantified using a BCA protein assay kit (#K819; BioVision, USA). For immunoblotting, proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% polyacrylamide gels and transferred onto a nitrocellulose membrane. Membranes were probed using primary antibodies, including anti-UCP-1 (#14670; Cell Signaling; 1:1000), PRDM16 (PA5-20872; Thermo Fisher Scientific; 1:1000), PGC-1a (#66369-I; Proteintech; 1:1500), Dio2 (ab77481; Abcam), Dio3 (ab233034; Abcam; 1:1000), VDAC (#4866; Cell Signaling; 1:1000), CtBP1 (ab14411; Abcam; 1:1000), TH (25859-1-AP; ProteinTech; 1:1000), β-actin (MA5-15739; Thermo Fisher Scientific; 1:1500) or β-tubulin (#179513; Abcam; 1:1500). Secondary antibodies, including anti-mouse IRDye 680 (C80926-17; 1:10,000) and anti-rabbit IRDye 800CW (C70918-03; 1:10,000), were purchased from LI-COR Bioscience (Lincoln, NE, USA). Signals were detected using infrared imaging (Odyssey, LI-COR Biosciences). Band intensity was quantified using Image Studio Lite 5.2 (Licor Biosciences).