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NBoxB reporter tethering assay HeLa cells were transiently transfected using PEI reagent (Polysciences) with 20 ng of RL (Renilla luciferase)5BoxB plasmid, 20 ng of control FL (Firefly luciferase) plasmid, and varying amounts of plasmids expressing NHAtagged proteins to obtain equal expression, as determined by SDSPAGE and analyzed by Western blotting, as described previously (Zipprich et al , 2009

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Countering opioid-induced respiratory depression in male rats with nicotinic acetylcholine receptor partial agonists varenicline and ABT 594