Journal: bioRxiv

Article Title: An examination of the divergent spatiotemporal signaling of GLP-1R versus GIPR in pancreatic beta cells

doi: 10.1101/2022.08.17.504231

Figure Lengend Snippet: ( A ) Western blot assessment of SNAP-GLP-1R or GIPR over tubulin levels in INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells with or without stimulation with 100 nM GLP-1 or GIP, respectively, for 6 hrs in the presence of the protein synthesis inhibitor cycloheximide; n =3. ( B ) Representative Western blot results from (A). Note that the top bands were used to quantify the SNAP-receptor levels, as they correspond to the glycosylated forms of the receptors, known to be biologically active and correctly inserted at the plasma membrane . ( C ) Percentage of GLP-1R vs GIPR, labelled with the cell-permeable SNAP-tag probe BG-OG, and corresponding representative images from INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells with or without stimulation with 100 nM GLP-1 or GIP, respectively, for the indicated times in the presence of cycloheximide; n =4. ( D ) Percentage of co-localization (Mander’s coefficient) and representative images of SNAP-GLP-1R vs -GIPR (labelled with SNAP-Surface 649) with Lysotracker Green in INS-1 832/3 SNAP-GLP-1R or SNAP-GIPR cells stimulated with 100 nM GLP-1 or GIP for 1 hr; n =5. Data are mean ± SEM, compared by ratio-paired or unpaired t-test or two-way ANOVA with Sidak’s post-hoc test; ***p<0.001, ****p<0.0001.

Article Snippet: INS-1 832/3 SNAP- GLP- 1R or SNAP-GIPR cells were labelled in suspension with 40 nM SNAP- Lumi4- Tb and 1 mM SNAP- Surface 649 (New England Biolabs, Hitchin, UK) for 1 hr at room temperature in complete medium.

Techniques: Western Blot

Journal: Nature Communications

Article Title: Multivalent interactions essential for lentiviral integrase function

doi: 10.1038/s41467-022-29928-8

Figure Lengend Snippet: a Schematic of the photobleaching experiment. Intasomes containing biotin- and Cy3-conjugated vDNA were immobilized on a streptavidin-coated cover slip (light blue rectangle). IN and vDNA are shown as gray and black cartoons, respectively; biotin and Cy3 are depicted as a purple circle and yellow solar symbol, respectively. Following incubation with LEDGF/p75 (red cartoons) conjugated with Surf649 (red solar symbol) and a wash with buffer containing 0.2, 0.5, or 1 M NaCl, immobilized intasomes were observed by TIRF microscopy. The individual steps of Surf649 photobleaching during illumination with a 640-nm laser (hν) were counted. b Representative images of surface attached intasome (vDNA-Cy3, yellow; LEDGF/p75-Surf649, red) molecules. The field of view of 14.4 by 14.4 μm shown here is representative of the dataset, which included three areas of 81.9 by 81.9 μm (see Methods section for details). Dotted, white line squares indicate individual intasome-LEDGF/p75 complexes. c Left: examples of stepwise photobleaching traces of LEDGF/p75-Surf649 at increasing NaCl concentration: 0.2 M (blue), 0.5 M (magenta), and 1.0 M (dark gray). The vertical axis represents fluorescence in arbitrary units (arb. units). Right: Box-and-whiskers plots summarizing statistical analysis of the number of LEDGF/p75-Surf649 photobleaching steps per intasome, for various NaCl concentrations (see Supplementary Table for further details). Each box encloses data between 25th and 75th percentiles, with the median value displayed as a horizontal line. Vertical lines (whiskers) indicate 10th and 90th percentiles; outliers are indicated with closed circles. Source data are provided as a Source Data file.

Article Snippet: LEGDF-SNAPf protein was labeled with SNAP-Surface649 (Surf649, New England Biolabs).

Techniques: Incubation, Microscopy, Concentration Assay, Fluorescence