Live cells were imaged 48 h posttransfection in KR2 buffer on a Nikon inverted epifluorescence selleck microscope with a charge-coupled device camera. About 12�C15 cells were found to express Epac-camps, and 6�C10 cells/35 mm glass bottom dish were found to express both fluorescent proteins, indicating the ��-cells. Pancreatic ��-cells from multiple 35-mm dishes were selected, and fluorescence was recorded before and during stimulation with 100 nM exendin-4. The FRET ratio was recorded every 5 s. Dynamic changes in cAMP estimated from the change in FRET ratio was acquired by MetaFluor software (Universal Imaging). GLP-1R-cyan fluorescent protein and yellow fluorescent protein-SUMO-1 live cell FRET.

MIN6 cells were transfected with cyan fluorescent protein (CFP)-GLP-1R and yellow fluorescent protein (YFP)-SUMO-1 or conjugation-deficient YFP-SUMO��GG construct where a stop codon was introduced at G96 to remove the last four amino acids essential for covalent interaction in SUMO-1. The cells were imaged on a live cell Olympus wide-field IX-50 fluorescent microscope. FRET was calculated by CFP (donor) bleaching, and the images were acquired every 200 ms. Fluorescence decay was calculated for the entire cell using ImageJ software (NIH). Time constants were calculated using Graphpad Prism software. Cell-Surface Biotinylation For cell-surface biotinylation, MIN6 cells cotransfected with untagged SUMO and GLP-1R-GFP were biotinylated on a 10-cm plate for 45 min and lysed in RIPA buffer. Biotinylated receptor was purified according to the manufacturer’s instructions (Thermo Fisher Scientific, Rockford, IL).

Biotinylated receptors at the membrane and nonbiotinylated receptors in the cytosol were detected by an anti-GFP monoclonal antibody (Roche Applied Science). Densitometric analysis was carried out using Image J software. Immunofluorescence MIN6 cells transfected with GLP-1R-HA and GFP-SUMO-1 were fixed in 4% PFA for 15 min, and permeabilization was not carried out to enable detection of only NH2-terminally HA epitope-tagged GLP-1R at the cell surface. The cells were incubated with rabbit HA antibody and stained with TRITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA). Immunostained and live cell images were acquired on a Leica TCS SP2 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) with sequential scanning of GFP and mCherry signals.

cAMP Assay MIN6 Dacomitinib cells were transfected with GFP or GFP-SUMO and sorted according to GFP fluorescence 48 h posttransfection. Cells expressing GFP or GFP-SUMO were plated in 96-well plates and treated with 100 nM exendin-4 for 3 h with and without 100 ��M IBMX. Quantitative analysis of cAMP was done using a sensitive chemiluminescence-based cAMP-specific enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions (Invitrogen).