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4 protocols using wwl70

1

Lipid Modification Proteomics Protocol

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Lipofectamine 2000, Lipofectamine RNAiMax, sodium dedocyl sulfate (SDS) solution, L-azidohomoalanine (L-AHA), Alexa Fluor 488-azide (AF488-az), Alexa Fluor 647-alkyne (AF647-alk), TRIzol reagent, and Prolong Gold Antifade Mountant with DAPI were purchased from Life Technologies (Burlington, ON). X-tremeGENE 9 was purchased from Roche (Indianapolis, IN). Palmostatin B was purchased from Merck Scientific (Billerica, MA). Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), Triton-X 100 (TX-100), sodium deoxycholate, CuSO4, palmitic acid, and 2-bromopalmitate were obtained from Sigma-Aldrich (St. Louis, MO). 17-ODYA, C75, WWL70, and RHC-80267 were purchased from Cayman Chemical (Ann Arbor, MI). HDFP, C83, and C115 were gifts from Dr. Brent Martin (University of Michigan), and FP-rhodamine was generously provided by Dr. Benjamin Cravatt (Scripps Institute).
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2

Analytical Reagents for Biochemical Assays

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AG, KT‐182, N‐oleylethanolamine‐d2 (OEA‐d2) and WWL70 were supplied by Cayman Chemicals (Ann Arbor, MI, USA) and 4‐MUH, 4‐MU, ACEA, butylated hydroxytoluene and zardaverine by Sigma‐Aldrich (Madrid, Spain). TOFA was supplied by Abcam (Cambridge, UK).
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3

Inhibition of ABHD6 and ABPP Assay

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KT182, an ABHD6 inhibitor, and HT-01, the activity-based protein profiling (ABPP) probe specific for ABHD6, were kindly provided by Drs. Hsu and Cravatt [26 (link)]. 2-Arachidonoylglycerol [glycerol-1,2,3-3H] was from American Radiolabeled Chemicals Inc. (Saint Louis, MO). Cyclooxygenase inhibitor assay kits including COX inhibitor and recombinant COX-1 or COX-2 activity assay kit were from Cayman Chemical (Ann Arbor, MI). siRNA (FlexiTube Mm_abhd6_3 and Allstars Negative Control) and HiPerfect transfection reagent were from QIAGEN (Valencia, CA). WWL70, methyl arachidonyl fluorophosphonate (MAFP), SR141716 (SR1), SR144528 (SR2), 2-AG, 2-AG-d8, and AA were purchased from Cayman Chemical. Other reagents including lipopolysaccharide (LPS) were purchased from Sigma-Aldrich (St. Louis, MO).
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4

Chemical Reagents for Receptor Studies

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The ABHD6 inhibitor WWL70, the CB1R antagonist AM281, and the CB2R antagonist AM630 were purchased from Cayman Chemicals (Ann Arbor, MI). All other chemicals and reagents were purchased from Sigma (St. Louis, MO), unless stated otherwise.
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