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For antibody labeling, after treatments at 37 C, cells were placed on ice to arrest further endocytosis before fixation, and the surface GLP-1R was detected by ELISA with monoclonal anti-human GLP-1R antibody 68 (Mab 3F52, Developmental Studies Hybridoma Bank (DSHB), 1/100) plus horseradish peroxidase (HRP)-conjugated rabbit anti-mouse secondary (ab97046, Abcam, 1/5,000)