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After 2 days of expression, cells were harvested in ice-cold PBS and subsequently lysed in AP-MS buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 10% Glycerol, 0.5 mM EDTA, 0.5% Nonidet P40) supplemented with protease inhibitor cocktail (Roche, Rotkreuz, Switzerland) by sonification, followed by centrifugation (20,000 g for 10 min
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This result may be explained by either the relative quality and quantity of food available or the fact that free-living birds cannot readily recover from the costs of an immune response because of the tradeoff between foraging and other energy demanding behaviors, such as mating and egg laying
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