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For Ube2d3-His 6 , the gene was cloned into pGEX-4T1 for expression as an N-terminal GST fusion with a TEV cleavage site

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Removal of the N-terminal 55 residues or ECD deletion completely abolished the activation effect of compound 2 despite that it binds to the cytoplasmic side of TM6, consistent with our previous report that ECD is required for GLP-1R and GCGR activation 34