Fig. 2: LUXendin645 binding, signaling, and labeling.
a Exendin4(9–39), S39C-Exendin4(9–39), and LUXendin645 (LUX645) display similar antagonistic properties (applied at 1 µM) in HEK293-SNAP_GLP1R following 30 min GLP-1-stimulation (n = 4 independent assays). b LUXendin645 weakly activates GLP1R in the presence of the positive allosteric modulator (PAM) BETP (25 µM) (30 min stimulation in HEK293-SNAP_GLP1R) (Ex4, +ve control) (n = 4 independent assays). c LUXendin645 labels AD293-SNAP_GLP1R cells with maximal labeling at 250–500 nM (n = 4 independent assays). d LUXendin645 signal cannot be detected in YFP-AD293 cells (scale bar = 212.5 µm) (n = 3 independent assays). e Representative confocal z-stack showing LUXendin645 staining in a live islet (n = 27 islets, six animals, three separate islet preparations) (scale bar = 37.5 µm). f As for e, but two-photon z-stack (scale bar = 37.5 µm) (representative image from n = 27 islets, seven animals, three separate islet preparations). g, h 250 nM LUXendin645 internalizes GLP1R in MIN6 β-cells when agonist activity is conferred using 25 µM BETP (Ex4 and Ex9 were applied at 100 and 250 nM, respectively) (scale bar = 21 µm) (representative images from n = 12 coverslips, three independent repeats) (one-way ANOVA with Bonferroni’s test; F = 217.6, DF = 3). i, j LUXendin645 signal co-localizes with a GLP1R monoclonal antibody in islets (n = 13 islets, three separate islet preparations) and MIN6 β-cells (representative images from n = 24 coverslips, three independent repeats) (scale bar = 26 µm). k LUXendin645 improves membrane visualization compared to antibody (scale bar = 12.5 µm). Representative images are shown, with ___location of intensity-over-distance measures indicated in blue (n = 18 islets, five animals, three separate islet preparations). l, m LUXendin645 co-localizes with Surface 488, pre-applied to Glp1r null SNAP_hGLP1R-INS1GLP1−/− cells l. Pre-treatment with Exendin4(1–39) to internalize the GLP1R reduces LUXendin645–labeling m (scale bar = 10 µm) (representative images from n = 3 independent repeats). LUXendin645 was applied to cells at 250 nM and tissue at 50–100 nM. GLP-1 glucagon-like peptide-1; Ex9 Exendin4(9–39); S39C S39C-Exendin4(9–39); Ex4 Exendin4(1–39). Mean ± s.e.m. are shown. **P < 0.01 for all statistical tests. Source data are provided as a Source Data file.
