This material is available free of charge via the Internet at xxxxxx. Publisher’s Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. In brief, the enzyme reaction was initiated by combining 1 nM rFAAH with 20 M of 14C-labeled oleamide in 500 L reaction buffer (125 mM TrisCl, 1 mM EDTA, 0.2% glycerol, 0.02% Triton X-100, 0.4 mM Hepes, pH 9.0) at room temp in the presence of three different concentrations of the inhibitor. The enzyme reaction was terminated by transferring 20 L of the reaction combination to 500 L of 0.1 N HCl at three different time points. The 14C-labeled oleamide (substrate) and oleic acid (product) were extracted with EtOAc and analyzed by TLC as detailed.13 The Ki of the inhibitor was calculated using a Dixon storyline as described.13 The purity of Aldosterone D8 each inhibitor (>95%) was determined on an Agilent 1100 LC/MS instrument on a ZORBAX SB-C18, 3.5 mm 50 mm, having a flow rate Gimap5 of 0.75 mL/min and detection at 220 and 254 nm, having a 10C98% acetonitrile/water/0.1% formic acid gradient and a 50C98% acetonitrile/water/0.1% formic acid gradient (see Assisting Information). Preparations of Mouse Cells Proteomes Tissues were Dounce-homogenized in PBS, pH 7.5, followed by a low-speed spin (1,400 g, 5 min) to remove debris. The supernatant was Aldosterone D8 then subjected to centrifugation (64,000 g, 45 min) to provide the cytosolic portion in the supernatant and membrane portion like a pellet. The pellet was washed and resuspended in PBS buffer by sonication. Total protein concentration in each portion was determined using a protein assay kit (Bio-Rad). ABPP Studies Cells proteomes, diluted to 1 1 mg/mL in PBS, were preincubated with inhibitors (10C10,000 nM, DMSO stocks) for 10 min and then treated with rhodamine-tagged fluorophosphonate (FP-rhodamine 100 nM, DMSO stock) at 25 C for 10 min. Reactions were quenched with SDS-PAGE loading buffer, subjected to SDS-PAGE, and visualized in-gel using a flatbed fluorescence scanner (MiraBio). Labeled proteins were quantified by measuring integrated band intensities (normalized for volume); control samples (DMSO alone) were regarded as 100% activity and inhibitor-treated samples were expressed as a percentage of remaining activity. IC50 ideals were identified from dose-response curves from three tests at each inhibitor Aldosterone D8 concentration using Prism software (GraphPad). Supplementary Material 01Click here to view.(469K, pdf) Acknowledgments We gratefully acknowledge the monetary support of the National Institutes of Health (Give DA015648, D.L.B.). We say thanks to Raj. K. Chadha for the X-ray crystal structure of (S)-54 and B. F. Cravatt for the supply of FAAH used in the enzymatic assays. Footnotes Assisting Information. Full experimental procedures, characterization and purities of the candidate inhibitors, and enzyme inhibition measurement standard deviations for Numbers 3, ?,55 and ?and77 Aldosterone D8 and Scheme 3. This material is available free of charge via the Internet at xxxxxx. Publisher’s Disclaimer: This is a PDF file of an unedited manuscript that has been approved for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the producing proof before it is published in its final citable form. Please note Aldosterone D8 that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain..