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Cells were washed with PBS 24 hours after transfection and imaged in Krebs-Ringer bicarbonate-Hepes (KRBH) buffer (140 mM NaCl, 3.6 mM KCl, 1.5 mM CaCl 2 , 0.5 mM MgSO 4 , 0.5 mM NaH 2 PO 4 , 2 mM NaHCO 3 , 10 mM Hepes, 0.1% BSA, saturated with 95% O 2 / 5% CO 2 , pH 7.4) supplemented with 11 mM glucose and imaged ratiometrically at excitation wavelengths of 405 and 488 nm and emission of 530 nm using a Zeiss LSM-780 inverted confocal laser-scanning microscope with a 63x oil immersion objective, with images taken every 6 seconds, in a 37°C heated chamber

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This ultimately leads to a much larger interaction pattern for GLP-2 (ranging from Asp 3P to Asn 16P ) than for GLP-1, where only two residues of GLP-1 interact with TM1