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Cell pellets were collected and resuspended in lysis buffer (50 mM Tris pH 8.0, 200 mM NaCl, 5 mM β-mercaptoethanol, 10% glycerol, and 1X Halt protease inhibitor (Thermo Fisher), and frozen overnight at −80 °C

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The pharmacology in animal studies and early human studies supported this design: AOD-9604 stimulates lipolysis and inhibits lipogenesis through activation of beta-3 adrenergic receptors in adipose tissue, without producing the IGF-1 elevation or insulin resistance signal that exogenous GH produces