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Insoluble material was removed by centrifugation at 30,000g for 20 min and the solubilized complex was immobilized by batch binding to M1 anti-Flag affinity resin in the presence of 5mM CaCl 2

The culture media was substituted by the media supplemented with 10% of Cell Count Reagent SF and incubated for 1 h

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5.1.2 Effects on pancreatic -cell glucagon release In addition to their insulinotropic effects on -cells, both GIP and GLP-1 play a role in regulating glucagon secretion of pancreatic -cells, with GIP increasing and GLP-1 suppressing glucagon secretion, both in a glucose-dependent manner