Lipidex 1000 resin

Docosahexaenoic acid (DHA), arachidonic acid (AA), oleic acid (OA), and all other common chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Three of the four representative cannabinoid ligands, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), CP55940 (2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol), and SR141716A (rimonabant; 5-(4-Chlor ophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide), were obtained from the National Institute on Drug Abuse (NIDA). The cannabinoid ligand WIN55212-2 ((R)-(+)-[2,3-Dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone) was purchased from Tocris Bioscience (R&D Systems, Minneapolis, MN, USA). The selective adipocyte FABP (FABP4) inhibitor, BMS309403 (2-[2′-(5-Ethyl-3,4-diphenyl-1H-pyrazol-1-yl)biphenyl-3-yloxy]-acetic acid), was purchased from EMD Chemicals (San Diego, CA, USA). The [9, 10-³H] oleic acid and Lipidex 1000 resin used for the radioactive competition binding assay were from Perkin Elmer (Waltham, MA, USA).

Pioglitazone (Pio; Cayman Chemical) and 1-hydroxyPioglitazone (PioOH; Axon Medchem) were purchased from commercial sources. 100% purity for PioOH was validated by the supplier by HPLC, NMR, and MS. Pio purity was also validated by the supplier: ≥ 98% by HPLC. Pio and PioOH were prepared in DMSO-d₆ as 50 mM and 7 mM stocks, respectively. Human PPARγ LBD (residues 203–477, isoform 1 numbering) was expressed in Escherichia coli BL21(DE3) cells as TEV-cleavable hexahistidine (6xHis)-tagged fusion protein using protocols previously described ^{11 (link), 12 (link)}. Purified protein was delipidated using LIPIDEX 1000 resin (Perkin Elmer) and stored in 20 mM potassium phosphate (pH 7.4), 50 mM potassium chloride, 0.5 mM EDTA, and 5 mM TCEP. FITC-labeled peptides derived from the TRAP220 coactivator (residues 638–656; NTKNHPMLMNLLKDNPAQD) and NCoR1 corepressor (residues 2256–2278; DPASNLGLEDIIRKALMGSFDDK) were synthesized by Lifetein; peptides contained a six-carbon linker (Ahx) after the FITC label, and the C terminus was amidated for stability.