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Proteins were separated by SDSPAGE, transferred to PVDF membranes (Millipore) and probed with primary antibodies in 1 TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl and 0.1% Tween-20) with 5% fat-free milk at 4 C overnight

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Microendoscopic calcium imaging revealed morphine inhibits the PVN Glu DMV Ach small intestine circuit, and this is accompanied by decreased small intestinal motility