Radioimmunoprecipitation buffer

Organ-specific T4, T3, and TRIAC contents were measured using LC-MS/MS.¹⁸ (link) We mechanically homogenized 50 mg of either the liver or cerebrum of a mouse in 300 μL radioimmunoprecipitation buffer (Nacalai Tesque) and left the homogenate on ice for 30 min. Supernatants were centrifuged at 10,000 g for 10 min at 4°C and collected in microtubes. We added 300 μL methanol and 600 μL chloroform, mixed with vortex mixer, and centrifuged at 15,000 g for 2 min at 4°C. The upper water/methanol phase was collected in new microtubes. We injected 6 μL of each sample into a Triple Quad 5500+ system and QTRAP Ready (AB SCIEX). Chromatography was performed using InertSustain C18 column (2.1 × 150 mm, 5 μm; GL Sciences, Tokyo, Japan) maintained at 40°C. A gradient of mobile phase A (0.5 mM ammonium fluoride in water) and mobile phase B (methanol) was used. The general conditions were as follows: 40% methanol (0–1 min), 40–90% methanol linear gradient (1–10 min), 90% linear gradient (10–15 min); flow rate of 0.2 mL/min. The following MS settings were adopted: curtain and collision gas pressure of 40 and 8 psi, respectively; ion spray voltage of −4500 V; temperature of 500°C; and ion source gas 1 and 2 pressure of 80 and 70 psi; correspondingly. T4, T3, and TRIAC were all measured with ESI in negative mode by MRM. The limits of quantification of T4, T3, and TRIAC were all 0.01 ng/mL.

To analyze protein expression, frozen aortic samples were pulverized using the SK Mill (TOKKEN, Japan) and proteins extracted with radioimmunoprecipitation buffer (No. 08714-04; Nacalai Tesque, Japan). Immunoblotting was performed with antibodies as indicated in the Major Resources Table in the online-only Data Supplement. Plasma levels of IL-17A were measured using a bead-based assay (Bio-Plex, No. 171-G5013M; Bio-Rad). To analyze mRNA expression, we isolated total RNA from the same part of the aorta as the protein expression analysis using the RNeasy kit (Qiagen). We performed transcriptome analyses using the SurePrint G3 Mouse Gene Expression v2 8x60K Microarray Kit (Agilent). The dataset has been deposited to the Gene Expression Omnibus of the National Center for Biotechnology Information (accession No. GSE116434). Biological process–focused gene enrichment analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (https://david.ncifcrf.gov/)^{18 (link)} with the Gene Ontology terms set to GOTERM_BP_FAT. Expression of the indicated genes was measured by quantitative reverse transcription polymerase chain reaction using commercially available probes (Qiagen).