Journal: bioRxiv

Article Title: Differential recognition of opioid analgesics by µ opioid receptors: Predicted interaction patterns correlate with ligand-specific voltage sensitivity

doi: 10.1101/2021.12.02.470941

Figure Lengend Snippet: (A) Representative FRET-based single cell recording of MOR-induced Gα i activation under voltage clamp conditions used for fit in and S9c, the voltage protocol indicated below (mean ± SEM, n=4). (B) Representative FRET-based single cell recording of MOR-induced Gα o activation under voltage clamp conditions used for fit in c, the voltage protocol indicated below (mean ± SEM, n=7). (C) Voltage dependence of naloxone-induced Gα i activation (blue) compared to Gα o activation (magenta). Activation was determined by clamping the membrane from -90 mV to different potentials and plotted relatively to 0 mV. Data was fitted to Boltzmann function resulting in z-factor of 1.17 for Gα i and 1.2 for Gα o and a V 50 - value of 31 mV for Gα i and 27 mV for Gα o . (D) Average FRET-based single cell recording of arrestin-mTur2 interaction with MOR-sYFP2 under voltage clamp conditions, the voltage protocol indicated below (mean ± SEM, n=7). (E) Average FRET-based single cell recording of arrestin-mTur2 interaction with MOR-sYFP2 induced by the weak partial agonist tramadol (mean ± SEM, n=5).

Article Snippet: Gß-2A-cpV-Gy 2 -IRES-Gα i2 - mTur2 was purchased from Addgene (Watertown, Massachusetts, USA, plasmid #69624 ( )).

Techniques: Activation Assay, Membrane

Journal: Analytical and bioanalytical chemistry

Article Title: Optical approaches for single cell and subcellular analysis of GPCR-G protein signaling

doi: 10.1007/s00216-019-01774-6

Figure Lengend Snippet: FRET and BRET based biosensors

Article Snippet: Therefore, appropriate FRET or BRET probes can be designed to examine intermolecular receptor-G proteins and G protein-G protein interactions, thus measuring GPCR and G protein activation in real-time [ 126 , 127 , 122 ]. table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Biosensor Name Target Number of molecules Physical type Pubmed ID Addgene plasmid # Gαi3 v1 Gαi Bimolecular or other FRET 16371464 Gαi-Gβ1 Gαi Bimolecular or other FRET 14673086 Gαi-Gγ2 Gαi Unimolecular FRET 14673086 Gαi2 Sensor v2 Gαi Bimolecular or other FRET 26799488 #69624 Gαi1 Sensor v2 Gαi Bimolecular or other FRET 26799488 #69623 RLucll-117-Gαs + GFP10-GY1 Gαs Bimolecular or other BRET 27499021 Gαs Sensor Gαs Bimolecular or other FRET 16963443 Gαq Sensor (v2) Gαq Bimolecular or other FRET 21619590 Gα13 Sensor (v2) Gα13 Unimolecular FRET 29505611 #112933 HCN2-camps cAMP Unimolecular FRET 17038640 Epac1-camps cAMP Unimolecular FRET 15231839 Epac2-camps cAMP Unimolecular FRET 15231839 mICNBD-FRET cAMP Unimolecular FRET 27003291 CAMYEL cAMP Unimolecular BRET 17283075 BFP-PMCA-GFP Ca 2+ Unimolecular FRET 17901055 AKAR4 PKA Unimolecular FRET #61619 CKAR PKC Unimolecular FRET 12782683 #14860 Open in a separate window FRET and BRET based biosensors 5.1.

Techniques: Plasmid Preparation

Journal: Antibodies

Article Title: Cross-Reactivity of N6AMT1 Antibodies with Aurora Kinase A: An Example of Antibody-Specific Non-Specificity

doi: 10.3390/antib13020033

Figure Lengend Snippet: Antibodies against N6AMT1.

Article Snippet: Samples were blocked with 3% Bovine Serum Albumin (BSA)/PBS solution for 1 h at RT and stained with anti-N6AMT1 (1:100, CQA1550, Cohesion Biosciences, London, UK), anti-N6AMT1 (1:100, HPA059242, Atlas antibodies, Bromma, Sweden), anti-N6AMT1 (1:100, 16211-1-AP, Proteintech, Rosemont, IL, USA), anti-N6AMT1 (1:100, PA5-121076, Invitrogen, Waltham, MA, USA), anti-N6AMT1 (1:100, ARP45845_P050, Aviva Systems Biology, San Diego, CA, USA), anti-α-tubulin (1:5000, T5168, Sigma-Aldrich, St. Louis, MO, USA), and pericentrin (1:1000, ab28144, Abcam, Cambridge, UK) diluted in 3% BSA/PBS solution, followed by three washes with PBS, and incubation with secondary anti-mouse and anti-rabbit antibodies conjugated to Alexa Fluor 568 or 488 (1:1000, Invitrogen, Carlsbad, CA, USA) diluted in 3% BSA/PBS solution.

Techniques:

Journal: British Journal of Pharmacology

Article Title: The G‐protein‐biased agents PZM21 and TRV130 are partial agonists of μ‐opioid receptor‐mediated signalling to ion channels

doi: 10.1111/bph.14702

Figure Lengend Snippet: Dissociation of Gα and Gβγ evoked by μ receptor (μOR) agonists in FRET experiments. Fluorescence measurements were performed in HEK293 cells transfected with mTurqoise2‐tagged Gαi1 (FRET donor), a Venus‐tagged Gγ2 (FRET acceptor), and Gβ1 expressed from a tricistronic vector (Gβ‐2A‐cpV‐Gγ2‐IRES‐Gαi1‐mTq2) and μ receptor as described in Section 2. (a) Upper panel, fluorescence traces detected at 480 for FRET donor (blue line) and 535 nm for FRET acceptor (yellow line); excitation wavelength was 430 nm. Lower panel shows the ratio of the individual traces (FRET signal). The applications of 10‐nM, 100‐nM, and 1‐µM DAMGO are indicated by the horizontal lines. (b) Summary of the data. (c) Representative measurement showing the effect of 1‐μM DAMGO and 1‐μM PZM21. (d) Summary of the normalized responses of FRET signals evoked by the applications of 1‐μM TRV130 (n = 6 cells), 1‐μM herkinorin (n = 5 cells), 10‐μM herkinorin (n = 7 cells), and 1‐μM PZM21 (n = 9 cells), compared to the effect of 1‐μM DAMGO in the same experiment for each compound. All fluorescence ratio traces were normalized to the ratio level before the application of the stimulus. Statistical significance was calculated using t test comparing the effect of DAMGO to the tested agonist in each experiment *P < .05

Article Snippet: Briefly, HEK293 cells were co‐transfected with the mTurqoise2‐tagged G αi1 (FRET donor), a Venus‐tagged G γ2 (FRET acceptor), and G β1 expressed from a tricistronic vector (Gβ‐2A‐cpV‐Gγ2‐IRES‐Gαi1‐mTq2; Addgene, catalogue # 69623; van Unen et al., 2016 ) and μ receptors.

Techniques: Fluorescence, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Live Cell Multicolour Lifetime Imaging Using Genetically Encodable Fluorophores

doi: 10.1101/2022.10.06.511114

Figure Lengend Snippet: (a) Live cells (middle panels) expressing a) tandem mTFP1-linker-mTurquoise2 (mTQ2), b) bi-cistronic mTFP1-T2A-mTQ2 and c) co-expression of mTFP1 and mTQ2 were tested to unmix both populations of plasmids. Scale bar 5 μm. Bar charts (right panels) plotting the lifetimes of mTFP1 (dark blue, 2.8 ns) and mTQ2 (cyan, 4.12 ns) when expressed alone were employed as a reference as explained in methods. The percentage obtained for the three cases depicted were 52+/−3%, n = 10; 49+/−4, n = 10 and 50+/−4%, n = 10; respectively. (b) A biological example shows live cells mimicking the HIV-1 virological synapse co-expressing CD4-mTFP1 and CXCR4-mTQ2 (target cells) and HXB2 Env and Gag-mCherry (effector cells). Intensity (left panel) and FLIM images (middle and right micrographs) are depicted showing that one can separate spectrally similar fluorescent proteins. Scale bar 5 μm. Bar diagrams are shown with quantification of the proportion of components colocalizing without Gag-mCherry (bottom left panels) and with Gag-mCherry (bottom right panels). Proportions of CD4-mTFP1 (60+/−10%, n = 5) and coreceptor CXCR4-mTQ2 (40+/−10%, n = 5) differ as compared to other regions where these receptors diffuse freely.

Article Snippet: Most of the DNA plasmids used in this article were obtained via Addgene ( https://www.addgene.org/ ; see and ). hCXCR4 and hCCR5 were cloned into pmTurquoise2-C1 (#60560) vector by ligating NheI/AgeI fragments into the corresponding sites of the vector, to make hCXCR4-mTQ2 and hCCR5-mTQ2.

Techniques: Expressing

Journal: bioRxiv

Article Title: Live Cell Multicolour Lifetime Imaging Using Genetically Encodable Fluorophores

doi: 10.1101/2022.10.06.511114

Figure Lengend Snippet: Cartoon showing cells co-cultured together co-expressing nine different FP plasmids (three by each cell). The cells were mixed after co-transfection of three different plasmids in each of the three initial wells. (b) The micrographs of live cells expressing nine different plasmids in three different spectral channels are shown. In the blue channel one has cells expressing histone H4 H4-mTFP1 and histone H2B-mTQ2. In the green channel there are cells expressing LaminB-mWasabi, cytosolic mAmetrine and EMTB-EGFP. In the red channel one has cells expressing cytosolic LSSmOrange, Mito-LSSmKate2, Alphactinin-mCherry and Cytosolic mKate2. Scale bar 10 μm. (c) the Intensity micrographs (left columns) together with the overall lifetime images (middle panels) and lifetime unmixed panels (right panels) are depicted. The corresponding pixel lifetime histograms before and after unmixing are also shown. The nine different fluorescent species were separated successfully.

Article Snippet: Most of the DNA plasmids used in this article were obtained via Addgene ( https://www.addgene.org/ ; see and ). hCXCR4 and hCCR5 were cloned into pmTurquoise2-C1 (#60560) vector by ligating NheI/AgeI fragments into the corresponding sites of the vector, to make hCXCR4-mTQ2 and hCCR5-mTQ2.

Techniques: Cell Culture, Expressing, Cotransfection