Journal: PLoS ONE

Article Title: Exploiting Ligand-Protein Conjugates to Monitor Ligand-Receptor Interactions

doi: 10.1371/journal.pone.0037598

Figure Lengend Snippet: (A) Scheme of the pulldown assay. The BG-ligand is immobilized on glutathione sepharose beads via GST-SNAP. Bound receptor proteins are concentrated on the beads after incubation with the extract and washing. (B) SNAP-eDHFR WT labeled with BG-647 (1 µM) was subjected to pulldown assay in the absence or presence of 100 µM NADPH using MTX immobilized on beads (MTX-BG +) or mock beads (MTX-BG -). Bound proteins were eluted with glutathione, submitted to SDS-PAGE and detected by in-gel fluorescence scanning. (C) Fluorescence signal of bound proteins normalized with the input signal in each gel (Mean±SD, n = 3). # represents P = 0.03 in paired t-test.

Article Snippet: SNAP-Cell Fluorescein (BG-fluorescein), SNAP-Cell TMR-Star (BG-tetramethylrhodamine), SNAP-Surface 647 (BG-647) were provided by NEB.

Techniques: Incubation, Labeling, SDS Page, Fluorescence

Journal: PLoS ONE

Article Title: Exploiting Ligand-Protein Conjugates to Monitor Ligand-Receptor Interactions

doi: 10.1371/journal.pone.0037598

Figure Lengend Snippet: (A) Scheme of the S-CROSS assay. SNAP-receptor and ligand-SNAP-CLIP are incubated to form a complex. BG-fluorophore-BC preferentially crosslinks proteins that are in close spatial proximity. (B) SNAP-eDHFR WT (1 µM) was mixed with MTX-SNAP-CLIP or unlabeled SNAP-CLIP (1 µM) in the presence or absence of free MTX (50 µM). The experiments were performed in the absence or presence of 100 µM NADPH. Then, the mixture was treated with 2.5 µM BG-647-BC. Labeled proteins were resolved by SDS-PAGE and detected by in-gel fluorescence scanning. (C) Fluorescence signal of the hetero-crosslinking products in the presence or absence of free MTX (Mean±SD, n = 3–5). # represents P = 0.001 in paired t-test. (D) S-CROSS assay in cell extract. SNAP-ORP7 was expressed in HEK293 cells. After preparation of extract, SNAP-ORP7 was subjected to S-CROSS assay using erlotinib-SNAP-CLIP (2 µM) in the absence or presence of free erlotinib (10 µM).

Article Snippet: SNAP-Cell Fluorescein (BG-fluorescein), SNAP-Cell TMR-Star (BG-tetramethylrhodamine), SNAP-Surface 647 (BG-647) were provided by NEB.

Techniques: Incubation, Labeling, SDS Page, Fluorescence

Journal: bioRxiv

Article Title: Commitment in the Pink1/Parkin mitophagy decision circuit

doi: 10.1101/2022.10.07.510659

Figure Lengend Snippet: (A) Cumulative distribution of single-cell Pink1 expression levels following 48h doxycycline treatment. (B) Representative images showing population-level expression levels. Scale bar, 50um. (C) Top: Representative cell showing colocalization between MtTether-SNAP stained with SNAP-647 and mitochondria immunostained for Tom20. Bottom: pixel intensity values along white line in the single-channel images above. Scale bar in merged channel image, 20um. (D) Representative images showing mitochondrial loss following prolonged rapalog-driven Pink1-mNG and Parkin-mKO2 recruitment. Asterisk indicates cell of interest. Dotted line in bottom right image indicates cell boundary. Scale bars, 10um. (E) Western blot comparison of phosphorylated S6 (top pair) to unphosphorylated S6 (bottom pair) following treatment with either rapamycin or rapalog.

Article Snippet: Prior to FACS, Pink1-mNG expression was induced with 50ng/mL Doxycycline Hydrochloride (Sigma-Aldrich, D3072) for 48 hours and MtTether-SNAP was stained with 1:2000 SNAP-647 SiR (NEB, s9102) for 30 minutes.

Techniques: Expressing, Staining, Western Blot

Journal: STAR Protocols

Article Title: SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos

doi: 10.1016/j.xpro.2022.101622

Figure Lengend Snippet: SNAP-tagged proteins can be visualized in both wild type (WT) and albino gastrula-stage Xenopus laevis embryos Images of WT (left) and Albino (right) Xenopus laevis embryos co-microinjected with E-cadherin-SNAPf mRNA (50 pg) and SNAP-Cell 647 SiR dye (10 μM). Scale Bars: 20 μm.

Article Snippet: SNAP-Cell 647-SiR , NEB , Cat #: S9102S.

Techniques:

Journal: STAR Protocols

Article Title: SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos

doi: 10.1016/j.xpro.2022.101622

Figure Lengend Snippet: Microinjecting or bathing embryos in dye can label E-cadherin-SNAPf in vivo (A) Images of Xenopus embryos expressing E-cadherin-SNAPf (50 pg) labeled via dye microinjection with SNAP-Cell 647 SiR (10 μM), SNAP-Cell TMR Star (25 μM), or SNAP-Cell Oregon Green (5 μM). (B) Images of Xenopus embryos expressing E-cadherin-SNAPf (50 pg) labeled via dye bath with SNAP-Cell 647 SiR (4 μM) or SNAP-Cell Oregon Green (3.3 μM). Scale Bars: 20 μm.

Article Snippet: SNAP-Cell 647-SiR , NEB , Cat #: S9102S.

Techniques: In Vivo, Expressing, Labeling

Journal: STAR Protocols

Article Title: SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos

doi: 10.1016/j.xpro.2022.101622

Figure Lengend Snippet: Microinjecting dye can label SNAPf-/Halo-Vinculin in vivo (A) Images of Xenopus embryos expressing Halo-Vinculin (10 pg) labeled via dye microinjection with Janelia Fluor 646 HaloTag (5 μM) and Janelia Fluor 549 HaloTag (5 μM). (B) Image of Xenopus embryo expressing SNAPf-Vinculin (10 pg) labeled via dye microinjection with SNAP-Cell 647 SiR (20 μM). Scale Bars: 20 μm.

Article Snippet: SNAP-Cell 647-SiR , NEB , Cat #: S9102S.

Techniques: In Vivo, Expressing, Labeling

Journal: STAR Protocols

Article Title: SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos

doi: 10.1016/j.xpro.2022.101622

Figure Lengend Snippet: SNAP- and Halo-tagged proteins can be visualized simultaneously, through dye microinjections or dye baths, in gastrula-stage Xenopus laevis embryos (A) Xenopus laevis gastrula-stage embryos expressing BFP-membrane (15 pg) (blue), Halo-ZO-1 (100 pg) labeled with microinjected Oregon Green HaloTag (10 μM) (green), and E-cadherin-SNAPf (50 pg) labeled with microinjected SNAP-Cell 647 SiR (5 μM) (red). (B) Xenopus laevis gastrula-stage embryos expressing BFP-membrane (15 pg) (blue), Halo-ZO-1 (100 pg) labeled via dye bath with Janelia Fluor 549 HaloTag (1.3 μM) (green), and E-cadherin-SNAPf (50 pg) labeled via dye bath with SNAP-Cell 647 SiR (4 μM) (red). Scale Bars: 20 μm.

Article Snippet: SNAP-Cell 647-SiR , NEB , Cat #: S9102S.

Techniques: Expressing, Labeling

Journal: STAR Protocols

Article Title: SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos

doi: 10.1016/j.xpro.2022.101622

Figure Lengend Snippet:

Article Snippet: SNAP-Cell 647-SiR , NEB , Cat #: S9102S.

Techniques: Recombinant, Software, Microscopy

Journal: STAR Protocols

Article Title: SNAP- and Halo-tagging and dye introduction protocol for live microscopy in Xenopus embryos

doi: 10.1016/j.xpro.2022.101622

Figure Lengend Snippet:

Article Snippet: SNAP-Cell 647-SiR , NEB , Cat #: S9102S.

Techniques: Recombinant, Software, Microscopy

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 ) Internalization AUC dose response curves from DERET assays in INS-1 832/3 SNAP-GLP-1R vs SNAP-GIPR cells stimulated with the indicated concentrations of GLP-1 or GIP, respectively. Results were fitted to a 3-parameter dose response curve to obtain Emax and logEC50 for both receptors, with comparisons between these parameters included; n =5. ( B ) Rates of GLP-1R vs GIPR internalization (k values) derived from (A) by one-phase association (with Y0=0) of baseline-deleted DERET data; n =5. ( C ) GLP-1R vs GIPR internalization dose response curves measured by high-content microscopy assay (HCA) in the same cells as above. Results fitted and Emax and logEC50 comparisons included as above; n =5. ( D ) Confocal microscopy analysis of SNAP-GLP-1R vs SNAP-GIPR (red) localization following 30 min stimulation with 100 nM GLP-1-FITC or GIP-FITC, respectively, in the same cells as above. ( E ) Confocal microscopy analysis of isolated intact mouse islets stimulated with fluorescently labelled agonists as indicated: WT islets were imaged following stimulation with 100 nM GLP-1-TMR, while both WT and GIPR -/- (KO) islets were imaged following stimulation with 1 µM GIP-TMR. ( F ) Quantification of surface GLP-1R vs GIPR levels in WT mouse islets using fluorescent agonist uptake data from (E); data corrected for binding affinity differences between both agonists; n =4. ( G ) GLP-1R vs GIPR recycling dose response curves measured by high-content microscopy assay (HCA) in cells from (C); n =5. ( H ) Confocal microscopy analysis of SNAP-GLP-1R vs SNAP-GIPR (red) co-localization with SNX27-GFP (green) following 3 hrs stimulation with 100 nM GLP-1 or GIP, respectively, in the same cells as above. Nuclei (DAPI), blue. Data are mean ± SEM, compared by paired or unpaired t-tests, or two-way ANOVA with Sidak’s post-hoc test; *p<0.05, **p<0.01, ***p<0.001; size bars: 10 µm.

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: Derivative Assay, Microscopy, Confocal Microscopy, Isolation, Binding Assay

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 ) Representative images from RICS analysis of GLP- 1R vs GIPR clustering, showing SNAP- Surface 488- labelled GLP-1Rs or GIPRs imaged in the basolateral plane of INS-1 832/3 SNAP-GLP-1R vs SNAP-GIPR cells after 5 min treatment in vehicle (Veh) and either 100 nM GLP-1 or GIP. Diffusion coefficients for individual ROIs are indicated on each image, with corresponding intensity traces, as well as 2D and fitted 3D autocorrelation maps for each ROI, are also depicted. ( B ) Average RICS diffusion coefficients for each receptor and treatment for each cell analyzed from n =4 experiments. ( C ) GLP- 1R clustering kinetics measured by TR- FRET in INS-1 832/3 SNAP-GLP-1R cells treated with 100 nM agonist as indicated, with vehicle- corrected AUCs included; n =4. ( D ) GIPR clustering kinetics measured by TR- FRET in INS-1 832/3 SNAP-GIPR cells treated with 100 nM agonist as indicated, with vehicle-corrected AUCs included; n =4. Data are mean ± SEM, compared by one-way ANOVA with Sidak’s test; ****p<0.0001; ns: non-significant.

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: Diffusion-based Assay

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: 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 ) GLP-1R vs GIPR plasma membrane activity dose response curves from bystander NanoBiT signaling assays performed in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Nb37-SmBiT, LgBiT-CAAX and SNAP-GLP-1R or -GIPR, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; results were fitted to 3-parameter dose response curves to obtain plasma membrane Emax and logEC50 for both receptors, with comparisons between these parameters included; n =4. ( B ) As for (A) but for GLP-1R vs GIPR endosomal activity in INS-1 832/3 GLP-1R KO vs GIPR KO cells transiently expressing Nb37-SmBiT, Endofin-LgBiT and SNAP-GLP-1R or -GIPR, after stimulation with the indicated concentrations of GLP-1 or GIP, respectively; n =4. Data are mean ± SEM, compared by paired t-tests; *p<0.05, ***p<0.001.

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: Activity Assay, Expressing