Snap surface 649

The assay was performed as previously described [20] (link). Cells were dual labelled with SNAP-Lumi4-Tb (40 nM) and 500 nM SNAP-Surface-649 (New England Biolabs, Hitchin, UK) for 30 min at 37 °C, in complete medium. After washing, labelled cells were resuspended in HBSS. TR-FRET signals at baseline and serially after agonist addition were recorded at 37 °C using a Spectramax i3x plate reader with HTRF module. GLP-1R clustering was quantified as the ratio of the fluorescence signal at 665 nm to that at 616 nm.

INS-1 832/3 EV and g1-2 cells transiently expressing the SNAP-GLP-1R were labeled at 37°C with 1 μM SNAP-Surface 649 fluorescent substrate (S9159S, New England Biolabs) in full medium before treatment with 100 nM exendin-4 or vehicle for 3 hours. Five minutes before the end of the latter incubation period, 1 μM LysoTracker Red DND-99 (L7528, Thermo Fisher Scientific) was added. The cells were washed in PBS and fixed in 4% PFA, mounted in ProLong Diamond antifade reagent with 4´,6-diamidino-2-phenylindole (Life Technologies), and imaged by confocal microscopy with an Imperial College London NHLI FILM Zeiss LSM-780 inverted confocal laser-scanning microscope and a 63×/1.4 numerical aperture oil immersion objective equipped with Zen software (ZEN 2.3 SP1, black, 64-bit, Carl Zeiss). Images were analyzed using ImageJ v1.53c. The Coloc 2 plugin was used for colocalization analysis.

We incubated 5 μM SNAP-CST with 10 μM SNAP-surface-649 (New England Biolabs) in 50 μL buffer C (35 mM Tris-HCl pH 7.5, 150 mM KCl, 10% glycerol, 0.01% Igepal, 1 mM 2-mercaptoethanol) for 16 h at 4 °C. Then we used an Amicon Ultra-4 10K centrifugal tube (Millipore) to remove the excess fluorescent dye and to concentrate the labeled CST complex. The labeled CST complex was divided into small aliquots and stored at −80 °C.

Two different fluorescent probes, SNAP-Surface 649 (red) and SNAP-Surface Alexa Fluor 488 (green; New England Biolabs), were used to label SNAP-α. All labeling reactions were carried out using a twofold molar excess of dye with 27 μM SNAP-α in 1 ml of 50 mM Tris-HCl pH 7.6, 2 mM dithiothreitol, 100 mM NaCl, 5% (v/v) glycerol (buffer Fα) for 2 hr at 23°C, followed by 6°C overnight with gentle rotation. Following the coupling, the reaction mixture was supplemented with 1 mM EDTA and excess dye was removed by gel filtration at 1 ml/min through a column (1.5 × 10 cm) of Sephadex G-25 (GE Healthcare, Chicago, IL) in buffer Fα+1 mM EDTA. Fractions containing the labeled SNAP-α were pooled and dialysed against 2 l of buffer Eα, frozen in liquid N₂ and stored in aliquots at –80°C. The degree of labeling was measured to be 90% for SNAP-α649 and 83% for SNAP-α488 by UV/vis spectrophotometry.

Calcium chloride (Cat. 10043-52-4), magnesium chloride (Cat. 7786-30-3), GABA (Cat. A2129), L-glutamate (Cat. 56-86-0), L-alanine (Cat. 56-41-7), L-trytophan (Cat. 73-22-3), neomycin (Cat. 1405-10-3) and spermine (Cat. 71-44-3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). NPS R-568 (Cat. 3815) and AC265347 (Cat. 6165) were purchased from Tocris Bioscience (Bristol, UK). NPS 2143 (Cat. ab145050) was obtained from Abcam (Cambridge, UK). Etelcalcetide (Cat. HY-P1955A) was from MedChemExpress (NJ, USA). Lipofectamine 2000 (Cat. 11668019) and Fluo4-AM (Cat. M14206) were supplied by Thermo Fisher Scientific (Waltham, MA, USA). SNAP-Surface 649 (Cat. S9159S) was from New England Biolabs (Ipswich, MA, USA). IP-One Gq kit (Cat. 62IPAPEB), BRET substrate coelenterazine h (Cat. S2001) and furimazine (Cat. N1120) were from Revvity (Codolet, France) and Promega (Madison, WI, USA), respectively.

Anti-FLAG-M2 affinity agarose gel and 3X FLAG peptide were obtained from Sigma Aldrich, n-dodecyl β-d-maltoside (DDM) was from Anatrace, protease inhibitor cocktail Set V was from Calbiochem/EMD Millipore Corp, Transporter 5™ transfection reagent (PEI MAX) was from Polysciences Inc., Antibiotic–Antimycotic (100X, containing 10,000 units penicillin, 10,000 μg/ml streptomycin and 25 μg/ml amphotericin) was from Gibco, and spin columns (product number 69705) were from Pierce Biotechnology. HEK293S GnTI^- cells were a kind gift of the Meyerson laboratory (Weill Cornell Medical College). NEBuilder HiFi DNA Assembly Cloning Kit, Phusion polymerase, Dpn1 and T4 DNA ligase were from New England Biolabs, and primers were obtained from IDT. Phospholipids (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)(POPG) and 1-palmitoyl-2-{6-[(7-nitro-2–1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC)) were obtained as stock solutions in chloroform from Avanti Polar Lipids. Sodium dithionite was from Sigma. SNAP-Surface 549 and SNAP-Surface 649 were from New England Biolabs. Bio-Beads SM2 resin was from Bio-Rad Laboratories; the resin was washed twice with methanol (25 ml per g of resin), twice with water and once with buffer (as indicated) prior to use.