plasmid pgex 4t2  (GE Healthcare)

Journal: bioRxiv

Article Title: Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

doi: 10.1101/2021.11.24.469908

Figure Lengend Snippet: ( A ) Bystander NanoBRET assay measuring miniG s -NLuc recruitment to the plasma membrane (with KRAS-Venus) vs endosomes (with Rab5-Venus) for 30 min in response to 100 nM GLP-1 in HEK293 SNAP-GLP-1R cells; n =5. ( B ) AUC quantification from (A). ( C ) Subcellular localisation of SNAP-GLP-1R (labelled with SNAP-Surface 549, red) after 1 min and 10 min of 100 nM GLP-1-FITC (green) stimulation in HEK293 SNAP-GLP-1R cells by confocal microscopy; nucleus (DAPI), blue; size bars as indicated. ( D ) Bystander NanoBRET assay of miniG s -NLuc recruitment to the plasma membrane as in (A) but in INS-1 832/3 SNAP-GLP-1R cells; n =4. AUC as indicated. ( E ) Time-lapse confocal microscopy analysis of SNAP-GLP-1R (labelled with SNAP-Surface 549, red) trafficking to Rab5-Venus (green)-positive endosomes in INS-1 832/3 SNAP-GLP-1R cells. Data is mean ± SEM; *p<0.05 by paired t-test.

Article Snippet: Transient transfections of Venus-, LgBiT- or NLuc-tagged mini-G constructs (all gifts from Professor Nevin Lambert, Augusta University, USA), Nb37-GFP (a gift from Professor Roshanak Irannejad, University of California San Francisco, USA), Gαs-YFP (Addgene plasmid #55781, a gift from Dr Catherine Berlot) and plasmids for the NanoBiT complementation and NanoBRET assays (see below) were performed using Lipofectamine 2000 (Thermo Fisher) for INS-1 832/3 and HEK293 cells according to the manufacturer’s instructions.

Techniques: Confocal Microscopy

Journal: bioRxiv

Article Title: Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

doi: 10.1101/2021.11.24.469908

Figure Lengend Snippet: ( A ) Representative images showing internalised SNAP f -GLP-1R after treatment with 100 nM GLP-1 for 30 min in HEK293 T-REx SNAP f -GLP-1R-SmBiT cells co-transfected with the indicated Venus construct. Residual surface label was then removed using Mesna. Phase contrast (Ph) and BG-SS-649 (SNAP; red) epifluorescence images are shown, along with segmented regions indicating Venus-expressing (green) and non-expressing (blue) cells. The white arrows highlight how mini-G s and mini-G q -expressing cells typically show reduced levels of internalised GLP-1R. ( B ) Quantification of experiment shown in (A), indicating average fluorescence of internalised GLP-1R in Venus-expressing and non-expressing cells treated with a range of concentrations of GLP-1; n =5 independent acquisitions. ( C ) Recruitment of each NLuc-tagged mini-G subtype to the plasma membrane in HEK293 T-REx SNAP f -GLP-1R-SmBiT cells co-transfected with KRAS-Venus and stimulated with GLP-1 for 30 min; n =5. ( D ) Time-lapse spinning disk images of SNAP-GLP-1R (labelled with SNAP-Surface 549, red) subcellular localisation from a representative mini-G s -Venus positive (green; white arrow) vs negative (yellow arrow) INS-1 832/3 SNAP-GLP-1R cell in response to 100 nM GLP-1 at the indicated frame times; size bars as indicated. ( E ) Percentage of GLP-1R internalisation in Venus-positive INS-1 832/3 SNAP-GLP-1R cells expressing each of the different mini-G subtypes tested (mini-G s , -G q and G i ) as well as empty mVenus as a control; n =3 independent time-lapse acquisitions. ( F ) AUC quantifications from (E). Data is mean ± SEM; **p<0.01 by one-way ANOVA with Dunnett’s test; ns (non-significant).

Article Snippet: Transient transfections of Venus-, LgBiT- or NLuc-tagged mini-G constructs (all gifts from Professor Nevin Lambert, Augusta University, USA), Nb37-GFP (a gift from Professor Roshanak Irannejad, University of California San Francisco, USA), Gαs-YFP (Addgene plasmid #55781, a gift from Dr Catherine Berlot) and plasmids for the NanoBiT complementation and NanoBRET assays (see below) were performed using Lipofectamine 2000 (Thermo Fisher) for INS-1 832/3 and HEK293 cells according to the manufacturer’s instructions.

Techniques: Transfection, Construct, Expressing, Fluorescence

Journal: bioRxiv

Article Title: Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

doi: 10.1101/2021.11.24.469908

Figure Lengend Snippet: ( A ) Clustering assay measured by TR-FRET in INS-1 832/3 SNAP-GLP-1R cells co-expressing mini-G s -LgBiT or pcDNA 3.1 as a control; n =5. ( B ) AUC quantification from (A). ( C ) Representative blots from detergent-resistant membrane (DRM) fractions collected from INS-1 832/3 SNAP-GLP-1R cells co-expressing mini-G s -Venus or mVenus as control under vehicle or 100 nM GLP-1-stimulated conditions. SNAP-GLP-1R detected with an anti-SNAP tag Ab and flotillin used as a DRM-specific loading control. ( D ) Quantification of SNAP-GLP-1R lipid raft recruitment from (C); n =5. ( E ) Lipid raft recruitment fold-increase to vehicle, calculated from (D). ( F ) Lipid raft-localised cAMP generation, dose responses to GLP-1 measured with AKAP79-CUTie in HEK293 T-REx SNAPf-GLP-1R-SmBiT cells co-expressing mini-Gs-LgBiT or pcDNA 3.1 as a control; logEC50 and Emax comparisons from the dose response curves displayed; n =6. Data is mean ± SEM; *p<0.05 and ns (non-significant) by paired t-test.

Article Snippet: Transient transfections of Venus-, LgBiT- or NLuc-tagged mini-G constructs (all gifts from Professor Nevin Lambert, Augusta University, USA), Nb37-GFP (a gift from Professor Roshanak Irannejad, University of California San Francisco, USA), Gαs-YFP (Addgene plasmid #55781, a gift from Dr Catherine Berlot) and plasmids for the NanoBiT complementation and NanoBRET assays (see below) were performed using Lipofectamine 2000 (Thermo Fisher) for INS-1 832/3 and HEK293 cells according to the manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

doi: 10.1101/2021.11.24.469908

Figure Lengend Snippet: ( A ) Percentage of GLP-1R internalisation in GFP-positive vs −negative INS-1 832/3 SNAP-GLP-1R cells expressing Nb37-GFP or an empty GFP vector as a control; n =4 independent experiments. ( B ) Bystander NanoBiT assay measuring Nb37-SmBiT recruitment to the plasma membrane (with CAAX-LgBiT) or to endosomes (with Endofin-LgBiT) for 30 min in response to 100 nM GLP-1 in HEK293 SNAP-GLP-1R cells; n =6. ( C ) AUC quantification from (B). ( D ) Subcellular localisation of Nb37-GFP (green) and SNAP-GLP-1R (labelled with SNAP-Surface 647, blue) to endosomes (labelled by co-expression of Rab5-mCherry, red) after 10 min stimulation with 100 nM GLP-1 in HEK293 SNAP-GLP-1R cells by confocal microscopy; size bars as indicated. Data is mean ± SEM; ns (non-significant) by one-way ANOVA with Dunnett’s test; *p<0.05 by paired t-test.

Article Snippet: Transient transfections of Venus-, LgBiT- or NLuc-tagged mini-G constructs (all gifts from Professor Nevin Lambert, Augusta University, USA), Nb37-GFP (a gift from Professor Roshanak Irannejad, University of California San Francisco, USA), Gαs-YFP (Addgene plasmid #55781, a gift from Dr Catherine Berlot) and plasmids for the NanoBiT complementation and NanoBRET assays (see below) were performed using Lipofectamine 2000 (Thermo Fisher) for INS-1 832/3 and HEK293 cells according to the manufacturer’s instructions.

Techniques: Expressing, Plasmid Preparation, Confocal Microscopy

Journal: bioRxiv

Article Title: Expression of mini-G proteins specifically halt cognate GPCR trafficking and intracellular signalling

doi: 10.1101/2021.11.24.469908

Figure Lengend Snippet: ( A ) Recruitment of each NLuc-tagged mini-G subtype to the plasma membrane in HEK293 cell lines stably expressing the indicated GPCR, co-transfected with KRAS-Venus and stimulated with the appropriate cognate agonist (100 nM GLP-1, 100 nM GIP, 1 mM proprionate, 100 nM isoproterenol, or 1 μM DAMGO) for 30 min; n =5. The NanoBRET signal has been adjusted to account for differences in receptor expression as measured by high content microscopy. ( B ) Impact on receptor internalisation of co-expression with the indicated mini-G-Venus fusion construct, determined by high content microscopy after 30 min stimulation with the appropriate cognate agonist as in (A); n =5. The absolute difference between “apparent percentage internalisation” (see Methods) in mini-G versus mVenus control cells is shown. ( C ) The association between mini-G subtype recruitment (shown in A) and receptor internalisation in Venus-expressing cells (same experiment as in B) for each GPCR. ( D ) Quantification of GIPR internalisation in Venus-positive vs −negative INS-1 832/3 SNAP-GIPR cells expressing mini-G s -Venus or empty mVenus as a control in response to 100 nM GIP; n =3 independent time-lapse acquisitions. ( E ) Quantification of β2AR internalisation in Venus-positive vs −negative HEK293 SNAP-β2AR cells expressing mini-G s -Venus or empty mVenus as a control in response to 100 nM isoproterenol; n =3 independent time-lapse acquisitions. ( F ) Quantification of FFA2R internalisation in Venus-positive vs −negative HEK293 T-REx SNAP-FFA2R cells expressing mini-G q -Venus or empty mVenus as a control in response to 1 mM proprionate; n =3 independent time-lapse acquisitions. ( G ) Quantification of μ-OR internalisation in Venus-positive vs −negative HEK293 SNAP-μ-OR cells expressing mini-G i -Venus or empty mVenus as a control in response to 1 μM DAMGO; n =3 independent time-lapse acquisitions. Data is mean ± SEM; **p<0.01 by one-way ANOVA with Dunnett’s test; ns (non-significant).

Article Snippet: Transient transfections of Venus-, LgBiT- or NLuc-tagged mini-G constructs (all gifts from Professor Nevin Lambert, Augusta University, USA), Nb37-GFP (a gift from Professor Roshanak Irannejad, University of California San Francisco, USA), Gαs-YFP (Addgene plasmid #55781, a gift from Dr Catherine Berlot) and plasmids for the NanoBiT complementation and NanoBRET assays (see below) were performed using Lipofectamine 2000 (Thermo Fisher) for INS-1 832/3 and HEK293 cells according to the manufacturer’s instructions.

Techniques: Stable Transfection, Expressing, Transfection, Microscopy, Construct