Supporting information for Ziouzenkova et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0538015100
Supporting Methods
Free Fatty Acid (FA) Content.
Free FA was determined with a standard enzymatic assay measuring total free FA content via spectrophotometry (Roche). This method was adapted for 96-well plates, with 300 µl as the total volume of media per well. For each assay, 82 µl of media (or standard) was combined with 18 µl of the commercial reaction mixture (solvent A and B) and used according to the manufacturer’s instructions and published reports, scaled to one-tenth the volume. FA levels were compared with a palmitic acid standard.
Scintillation Proximity Assay (SPA) Binding Assays.
Binding assays were done using known synthetic PPAR agonists obtained from Merck Research Laboratories. Human full-length human cDNA for PPARα and PPARδ were subcloned into pGEX-KT expression vectors, whereas pGEX-hPPARγ2 was constructed and used in the SPA assay as described (1). Two Merck Research Laboratory compounds, compound A {3-[4-(3-phenyl-7-propyl-benzisoxazole-6-yl)oxy]butyloxyphenylacetic acid} and compound B {3-chloro-4-[3-(3-trifluoromethyl-7-propyl-benzisoxazole-6-yl)oxypropyl]thiophenylacetic acid} were identified as PPAR ligands. [3H2]Compound A (5 nM) was used as the radioligand for PPARα and PPARγ, and [3H2]compound B (2.5 nM) was used for PPARδ. Results are expressed as the percentage inhibition, with an inhibitory constant (KiS) calculated by the equation of Cheng and Prusoff (2).1. Marelli-Berg, F. M., Peek, E., Lidington, E. A., Stauss, H. J. & Lechler, R. I. (2000) J. Immunol. Methods 244, 205–215.
2. Cheng, Y. & Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099–3108.