Journal: bioRxiv

Article Title: Chronic stress impairs autoinhibition in neurons of the locus coeruleus to increase asparagine endopeptidase activity

doi: 10.1101/2025.03.10.642335

Figure Lengend Snippet: ( A, B ) Confocal images of LC neurons showing immunoreactivities for TH and α2A-ARs, together with a merged one in non-stress and 3-day RS mice. Arrowheads indicate the membrane regions of TH-positive neurons, along which α2A-ARs were differentially expressed between the control and the RS mice. Asterisks indicate LC neurons. ( C ) Western blotting analyses showing the expression of α2A-ARs in non-stress and 3-day RS mice ( n = 6 and 7, respectively). Unpaired t -test, † p < 0.001. ( D, E ) Confocal images of LC neurons in non-stress and RS mice. Upper panels from left to right showing the respective immunoreactivity for TH, α2A-ARs (with rabbit anti-α2A-AR) and Na + -K + -pump and an enlarged image of the region enclosed with a rectangle (arrowheads) in its immediate left panel. Lower panels from left to right showing the merged image of TH and α2A-ARs, that of TH, α2A-ARs and Na + -K + -pump, that of α2A-ARs and Na + -K + -pump and an enlarged image of the region enclosed with a rectangle in its immediate left panel. ( F, G ) Western blotting analyses showing expressions of α2A-ARs and Na + -K + -pump in membrane fraction and those of α2A-ARs and β-actin in cytosol fraction in non-stress and 5-day RS mice ( n = 3 and 3 samples, respectively). Each sample represents the analysis result in the LC tissues obtained from 2-3 mice. Membrane; unpaired t -test, † p = 0.016. Cytosol; unpaired t -test, † p = 0.083.

Article Snippet: To examine the distribution of α2A-AR in LC neurons, mouse anti-TH (1:500; sc-25269, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-sodium potassium ATPase (1:1000; ab76020, Abcam, Cambridge, MA, USA) and goat anti-α2A-AR (1:200; ab45871, Abcam) or rabbit anti-α2A-AR (1:500; RA14110, Neuromics, Northfield, MN, USA) were used as primary antibodies, while cy5 donkey anti-mouse IgG (1:500; cy-2500, Vector Laboratories, Burlingame, CA, USA), FITC donkey anti-rabbit IgG (1:500; 711-095-152, Jackson ImmunoResearch, West Grove, PA, USA) and Cy3 donkey anti-goat IgG (1:500; 706-165-147, Jackson ImmunoResearch) were used as secondary antibodies.

Techniques: Membrane, Control, Western Blot, Expressing

Journal: bioRxiv

Article Title: Chronic stress impairs autoinhibition in neurons of the locus coeruleus to increase asparagine endopeptidase activity

doi: 10.1101/2025.03.10.642335

Figure Lengend Snippet: ( A-E ) Representative traces of NA-induced GIRK-I obtained from LC neurons in non-stress and 1-day, 2-day, 3-day, and 5-day RS mice. ( F, G ) Amplitudes of inward and outward components of NA-induced GIRK-I obtained from LC neurons in non-stress and 1-day, 2-day, 3-day, and 5-day RS mice ( n = 8, 7, 5, 6 and 8, respectively) decreased with the increase in the period of RS, in a way that can be described by a saturation function (red interrupted lines). The saturation level ( a + b ) and the half saturation constant ( c ) were determined by fitting the saturation function, defined as y = a + ( b * x) / ( c + x), to the data points. The values of a , b and c for the inward component of GIRK-I were −151.1, 108, and 0.9, respectively, and those for the outward component of GIRK-I were 81.1, –67, and 1.6, respectively. Inward component: one-way ANOVA, p < 0.001, post hoc fisher’s PLSD, 1-day; # p < 0.001 vs Non-stress and * p < 0.05 vs 3-day and 5-day, 2-day; # p < 0.001 vs Non-stress, 3-day; # p < 0.001 vs Non-stress, 5-day; # p < 0.001 vs Non-stress. Outward component: one-way ANOVA, p < 0.001, post hoc fisher’s PLSD, 1-day; p = 0.004 vs Non-stress and * p < 0.05 vs 3-day and 5-day, 2-day; p = 0.015 vs Non-stress, 3-day; p < 0.001 vs Non-stress, 5-day; p < 0.001 vs Non-stress. ( H ) Relative expressions of α2A and α2C mRNAs, normalized to GAPDH in LC neurons ( n = 5). Paired t -test, ‡ p = 0.014. ( I ) Relative expressions of GIRK1, GIRK2, and GIRK3 mRNAs, normalized to GAPDH in LC neurons ( n = 5). One-way RM ANOVA, p < 0.001, post hoc fisher’s PLSD; * p = 0.008 for GIRK1 vs GIRK2, * p < 0.001 for GIRK1 vs GIRK3, * p = 0.008 for GIRK2 vs GIRK3. ( J, K ) Relative expressions of α2A and GIRK1 mRNAs, respectively, normalized to GAPDH in LC neurons in non-stress mice ( n = 8), and 3-day RS mice ( n = 6). α2A-AR: unpaired t -test, † p = 0.020; GIRK1: unpaired t -test, † p = 0.013. (L) Normalized relative expressions of GIRK2 mRNA in LC neurons in non-stress mice ( n = 8) and 3-day RS mice ( n = 6), normalized to the ratio of the mean value of the relative expressions of GIRK1 mRNA to that of GIRK2 mRNA in LC neurons in non-stress mice ( I ). Unpaired t -test, † p = 0.037.

Article Snippet: To examine the distribution of α2A-AR in LC neurons, mouse anti-TH (1:500; sc-25269, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-sodium potassium ATPase (1:1000; ab76020, Abcam, Cambridge, MA, USA) and goat anti-α2A-AR (1:200; ab45871, Abcam) or rabbit anti-α2A-AR (1:500; RA14110, Neuromics, Northfield, MN, USA) were used as primary antibodies, while cy5 donkey anti-mouse IgG (1:500; cy-2500, Vector Laboratories, Burlingame, CA, USA), FITC donkey anti-rabbit IgG (1:500; 711-095-152, Jackson ImmunoResearch, West Grove, PA, USA) and Cy3 donkey anti-goat IgG (1:500; 706-165-147, Jackson ImmunoResearch) were used as secondary antibodies.

Techniques:

Journal: bioRxiv

Article Title: Chronic stress impairs autoinhibition in neurons of the locus coeruleus to increase asparagine endopeptidase activity

doi: 10.1101/2025.03.10.642335

Figure Lengend Snippet: ( A, B ) Differential free concentrations of NA to be metabolized by MAO-A into DOPEGAL between control and stress conditions: Under the control condition ( A ), NA in the cytosol is mostly taken up into the cytoplasmic vesicles by vMAT2 (thick arrow 3), rather than being directly metabolized by MAO-A into DOPEGAL (thin interrupted arrow). Vesicular NA is released from cell bodies as autocrine following [Ca 2+ ] i increases caused by action potentials, and the released NA activates α2A-AR-coupled GIRK channels, causing autoinhibition. Subsequently, the autocrine released NA is slowly taken up into the cytosol of LC neurons by NAT after dissociation from α2A-ARs. Under the stress condition ( B ), an activation of CRF receptors in LC neurons by stress inhibits leak K + channels and increases firing activities in LC neurons, subsequently causing a larger [Ca 2+ ] i increase together with Ca 2+ -induced Ca 2+ release (CICR). Impairment of autoinhibiton due to Ca 2+ dependent internalization of α2A-ARs-coupled GIRK channels leads to the persistent excitation in LC neurons, which enhances autocrine release of NA (thick arrow 1). Subsequently, the excessively autocrine released NA is taken up directly and rapidly by NAT into the cytosol without binding to α2A-ARs (thick arrow 2). Such a facilitation of re-uptake of NA by NAT would increase active NA storage into cytoplasmic vesicles by vMAT2 (thick arrow 3), while the rate of NA leakage from cytoplasmic vesicles would also increase (arrow 4) due to a dynamic equilibrium in cytoplasmic vesicles between active NA storage into cytoplasmic vesicles and passive NA leakage from cytoplasmic vesicles. Subsequently, such an increase in the rate of NA leakage would result in an increase in a MAO-A metabolite, DOPEGAL and AEP, leading to a production of cleaved tau N368 fragment and an impairment of learning/memory.

Article Snippet: To examine the distribution of α2A-AR in LC neurons, mouse anti-TH (1:500; sc-25269, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-sodium potassium ATPase (1:1000; ab76020, Abcam, Cambridge, MA, USA) and goat anti-α2A-AR (1:200; ab45871, Abcam) or rabbit anti-α2A-AR (1:500; RA14110, Neuromics, Northfield, MN, USA) were used as primary antibodies, while cy5 donkey anti-mouse IgG (1:500; cy-2500, Vector Laboratories, Burlingame, CA, USA), FITC donkey anti-rabbit IgG (1:500; 711-095-152, Jackson ImmunoResearch, West Grove, PA, USA) and Cy3 donkey anti-goat IgG (1:500; 706-165-147, Jackson ImmunoResearch) were used as secondary antibodies.

Techniques: Control, Activation Assay, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Induces Angiogenesis in Progenitor and Mature Endothelial Cells In Vitro: Investigation of the Possible Role of Adenosine

doi: 10.3390/ijms252413343

Figure Lengend Snippet: The effects of the adenosine receptor A 2A antagonist SCH58621, adenosine receptor A 2B inverse agonist, and antagonist MRS1706 on capillary-like tube formation by ( A ) ECFCs and ( B ) HUVECs, and on spheroid sprout formation by ( C ) ECFCs and ( D ) HUVECs, induced by ticagrelor, adenosine, and their combination. The values represent the means ± SDs from at least 6 independent biological replicates each performed in 2 technical replicates. * p < 0.02 and ** p < 0.01 compared to ticagrelor, adenosine, or their combination in the absence of SCH58621 and MRS1706. # p < 0.05 compared to ticagrelor, adenosine, or their combination in the presence of either SCH58621 or MRS1706. HUVECs: human umbilical vein endothelial cells, ECFCs: endothelial colony-forming cells.

Article Snippet: Adenosine, DPCPX (A 1 AR antagonist), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-ac-LDL), and adenosine deaminase were purchased from Sigma-Aldrich (St. Louis, MO, USA), and MRS1220 (A 3 AR antagonist), SCH58621 (A 2A AR antagonist), and MRS1706 (A 2B AR inverse agonist and antagonist) were purchased from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Induces Angiogenesis in Progenitor and Mature Endothelial Cells In Vitro: Investigation of the Possible Role of Adenosine

doi: 10.3390/ijms252413343

Figure Lengend Snippet: Representative images of 2D capillary-like tube formation by ECFCs that were treated with ticagrelor, adenosine, and their combination in the presence of adenosine receptor A 2A antagonist SCH58621, adenosine receptor A 2B inverse agonist, and antagonist MRS1706. Scale bar 100 μΜ.

Article Snippet: Adenosine, DPCPX (A 1 AR antagonist), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-ac-LDL), and adenosine deaminase were purchased from Sigma-Aldrich (St. Louis, MO, USA), and MRS1220 (A 3 AR antagonist), SCH58621 (A 2A AR antagonist), and MRS1706 (A 2B AR inverse agonist and antagonist) were purchased from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Ticagrelor Induces Angiogenesis in Progenitor and Mature Endothelial Cells In Vitro: Investigation of the Possible Role of Adenosine

doi: 10.3390/ijms252413343

Figure Lengend Snippet: Representative images of 3D spheroid sprout formation by ECFCs that were treated with ticagrelor, adenosine, and their combination in the presence of adenosine receptor A 2A antagonist SCH58621, adenosine receptor A 2B inverse agonist, and antagonist MRS1706. Scale bar 200 μM.

Article Snippet: Adenosine, DPCPX (A 1 AR antagonist), 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine-labeled acetylated LDL (DiI-ac-LDL), and adenosine deaminase were purchased from Sigma-Aldrich (St. Louis, MO, USA), and MRS1220 (A 3 AR antagonist), SCH58621 (A 2A AR antagonist), and MRS1706 (A 2B AR inverse agonist and antagonist) were purchased from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Human A 2A AR in different membrane or membrane-mimetic environments and sample preparation workflow. (a) A 2A AR compared in three different environments in this study: (left) detergent micelles, (middle) lipid nanodiscs, and (right) lipid vesicles. (b) Schematic of the sample preparation workflow for preparing lipid vesicles containing 19 F-labeled human A 2A AR for solid-state MAS NMR experiments.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Membrane, Sample Prep, Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Characterization of lipid vesicles containing human A 2A AR and pharmacological validation of A 2A AR in vesicles. ( a and b ) Representative negative stain electron micrographs of unilameller vesicles composed of POPC and POPS (70:30 molar ratio) ( a ) without and ( b ) with reconstituted A 2A AR. ( c ) Dynamic light scattering measurements of the distribution of the sizes of vesicles composed of POPC and POPS (70:30 molar ratio) without (green) and with (purple) A 2A AR. ( d and e ) Pharmacological characterization of A 2A AR in lipid vesicles. ( d ) Saturation binding experiment with 3 H-ZM241385 and A 2A AR in lipid vesicles containing POPC and POPS (70:30 molar ratio). The reported B max value represents the mean and associated error is the s.e.m. from 3 independent trials done in triplicate. ( e ) Radioligand competition experiments. K D and K i values are reported for the antagonist ZM241385 and the agonist NECA, respectively. The reported error represents the s.e.m. from 3 independent trials done in triplicate.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Staining, Binding Assay

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Determination of the orientation of A 2A AR in lipid vesicles. ( a ) Schematic of the fluorescence-quenching assay used to quantify receptor orientation within vesicles. Green circles represent position C289 labeled with Cy3. Receptors with Cy3 covalently attached to position C289 facing toward the vesicle interior are labeled “A 2A AR inside”, while receptors with Cy3 facing away from the vesicle interior are labeled “A 2A AR outside”. Grey circles represent Cy3-labels that have been chemically quenched. ( b ) Quantitative comparison of the orientation of A 2A AR in vesicles made from POPC or POPC and POPS (70:30 molar ratio). The orientations “A 2A AR inside” and “A 2A AR outside” are as defined in ( a ). Error bars indicate the s.e.m. calculated from n≥3 independent experiments. Statistically significant values are illustrated as ***p<0.005 using a 2-tailed unpaired t-test.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Fluorescence, Labeling, Comparison

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: Fluorescence thermal melting profiles of the A 2A AR complex with the antagonist in three different membrane mimetics. ( a ) Schematic of the fluorescence thermal shift assay as applied to A 2A AR in lipid vesicles. The inactive fluorescent dye (grey stars) shows increased emission upon covalent attachment with cysteines that become solvent accessible upon protein unfolding (orange stars). ( b ) Representative thermal melting profiles for A 2A AR in DDM/CHS detergent micelles, lipid nanodics containing POPC and POPS (70:30 molar ratio), and lipid vesicles containing POPC and POPS (70:30 molar ratio). ( c ) The melting temperature for A 2A AR in each membrane mimetic is reported as the mean of three independent experiments ± s.e.m.

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Fluorescence, Membrane, Thermal Shift Assay, Solvent

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F MAS NMR-observed conformational equilibria of human A 2A AR in complex with an antagonist in lipid vesicles measured with different experimental parameters. ( a ) 1-dimensional 19 F-MAS spectra of A 2A AR[A289C TET ] in complex with the antagonist ZM241385 reconstituted into POPC/POPS lipid vesicles recorded with 105 kHz 1 H TPPM decoupling at three different MAS frequencies. ( b ) NMR spectra from (a) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 and P3. ( c ) 1-dimensional 19 F-MAS spectra of the same sample used to measure the data in (a) recorded with 1 H TPPM decoupling power set to one-quarter of the applied MAS frequency. ( d ) NMR spectra from (c) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 and P3. ( e and f ) ( e ) 1-dimensional 19 F-MAS spectra of the same sample used to measure the data in (a) recorded with no 1 H decoupling and ( f ) NMR spectra from ( e ) are shown superimposed with Lorentzian deconvolutions. ( g-j ) Linewidths measured for populations P1 and P3 for A 2A AR in ( g-i ) lipid vesicles or ( j ) lipid nanodiscs or detergent micelles. Components colored green are from free TET, consistent with earlier NMR studies (see text).

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F MAS NMR-observed conformational equilibria of human A 2A AR in complex with the agonist NECA in lipid vesicles measured with low power TPPM 1 H decoupling at several MAS frequencies. ( a ) 1-dimensional 19 F-MAS spectra of A 2A AR[A289C TET ] in complex with the agonist NECA reconstituted into POPC/POPS lipid vesicles recorded with three different MAS frequencies and 1 H TPPM decoupling at an applied 1 H power of one-quarter of the MAS frequency. ( b ) NMR spectra from (a) are shown superimposed with Lorentzian deconvolutions with the minimal number of components that provided a good fit, labeled P1 through P5. Components colored green are from free TET, consistent with earlier NMR studies (see text).

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Labeling

Journal: bioRxiv

Article Title: The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19 F-NMR

doi: 10.1101/2024.10.14.618237

Figure Lengend Snippet: 19 F-NMR systematic comparison of the conformational equilibria of antagonist-bound and agonist-bound human A 2A AR[A289C TET ] across three membrane or membrane-mimetic systems by solution NMR in (a) detergent micelles and (b) lipid nanodiscs and by MAS solid-state NMR in (c) lipid vesicles. Same color scheme as in and .

Article Snippet: Plasmids containing A 2A AR[A289C] were transformed into the BG12 strain of Pichia pastoris (Biogrammatics) via electroporation.

Techniques: Comparison, Membrane

Journal: iScience

Article Title: Monitoring GPCR conformation with GFP-inspired dyes

doi: 10.1016/j.isci.2024.110466

Figure Lengend Snippet: Structural and spectral changes of A 2A AR L225C -DyeC induced by various ligands (A) Schematic representation of structural changes caused by agonists and the allosteric modulator HMA in A 2A AR L225C -DyeC. Structures of the active and inactive A 2A AR are sketched from PDB IDs 6GDG and 3RFM , respectively. , Agonist binding results in an outward shift of the intracellular part of the TM6. The structural effects of the allosteric modulator HMA remain unknown. (B) Emission spectra of A 2A AR L225C -DyeC bound to antagonists (ZM241385 and SCH58261), agonists (NECA and adenosine), allosteric modulator (HMA), and in the apo state. (C) Variation of the integrated intensity of A 2A AR L225C -DyeC bound to different ligands. The integrated intensity is quantified as the area under the fluorescence emission spectrum from 430 to 700 nm. (D) Variation of the intensity ratio I 520 /I 460 for different ligands. Each condition in C and D was measured at least 9 times with protein from at least three independent purifications; each protein sample was mixed with the ligand independently. The data represent the mean ± SD. The protein concentration was maintained at 10 μM; all ligands were added at a saturating concentration of 100 μM. The significance level is given according to the ordinary one-way ANOVA with the post hoc Tukey HSD test: ∗∗ p < 0.005, ∗ p < 0.05, ns, not significant..

Article Snippet: Figure 4 Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations.

Techniques: Binding Assay, Fluorescence, Protein Concentration, Concentration Assay

Journal: iScience

Article Title: Monitoring GPCR conformation with GFP-inspired dyes

doi: 10.1016/j.isci.2024.110466

Figure Lengend Snippet: Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations. The A 2A AR L225C helices are labeled from TM1 to H8 (TM6 is colored in red), the G-protein binding site is labeled in the active state. (C and D) Positions of the dimethoxybenzene ring of the DyeC label in the active (C) and inactive (D) complexes throughout the unbiased (i.e., without any external forces applied) MD simulations shown every 0.1 ns as orange/yellow dots, respectively. Each system was simulated for 1,000 ns in two replicates. The positions of the lipid head groups are schematically indicated by the gray dotted line. (E) Autocorrelation functions (ACFs) calculated for a vector describing the DyeC label position in the unbiased simulations shown in (C) and (D). Higher values of ACF suggest slower reorientational dynamics of the label. Results for two replicates in the active/inactive states are shown in orange/yellow. (F) Correlation between fluorescence emission maximum of DyeC in different solvents and their partition coefficient, logP. The logP values were obtained from the PubChem/Chemeo databases , and provided in Table S1 . The inverse correlation implies that the translocation of DyeC into the region of the polar head groups of lipids, as observed in the simulations of the active state, leads to a red shift in the fluorescence emission maximum.

Article Snippet: Figure 4 Molecular dynamics simulations of A 2A AR L225C -DyeC (A and B) Isosurfaces (yellow) delineate the low-free-energy regions (at +25 kJ/mol level relative to the global free energy minimum) explored by the proximal carbon atom of the dimethoxybenzene ring of DyeC label in the active (A) and inactive (B) states in metadynamics simulations.

Techniques: Labeling, Protein Binding, Plasmid Preparation, Fluorescence, Translocation Assay

Journal: Nature Communications

Article Title: Specific pharmacological and G i/o protein responses of some native GPCRs in neurons

doi: 10.1038/s41467-024-46177-z

Figure Lengend Snippet: a Effect of the indicated α 2 AR agonists on the net BRET of the G i1 and G oA sensors in CGNs co-transfected with Gα i1 Nluc and Venus Gγ 2 or Gα oA Nluc and Venus Gγ 2 (amounts of cDNA as in Fig. ). b Effect of the indicated α 2 AR agonists on the net BRET of the G i1 and G oA sensors in HEK293 cells co-transfected with the mouse α 2A AR (10 ng, 20 ng or 50 ng), and Gβ 1, Venus Gγ 2 and Gα i1 Nluc or Gα oA Nluc as in Fig. . Saturating concentrations of brimonidine (10 μM), oxymetazoline (100 μM), xylazine (50 μM), clonidine (100 μM), tizanidine (100 μM) were used in ( a , b ). c Detection of α 2 AR in cell membranes of CGNs, mock-transfected HEK293 cells and HEK293 cells transfected with indicated amount of α 2 AR, by western blotting. Values are mean ± SEM normalized as fold of α 2 AR expression in CGNs from four independent experiments. d The pEC 50 of brimonidine in CGNs ( n = 3) and transfected HEK293 cells ( n = 4) with indicated amount of α 2 AR measured by G i1 or G oA sensor. e Percentage of change in BRET ratio between Gα Nluc and Venus Gγ 2 induced by brimonidine between Gα Nluc and Venus Gγ 2 in HEK293 cells ( n = 3) (α 2 AR: 15 ng/well per 96-well plate) or CGNs ( n = 4) for the indicated Gα i1 , Gα i2 , Gα i3 , Gα oA , Gα oB or Gα z sensors (amounts of cDNA as in Fig. ). Values are mean ± SEM from at least three biologically independent experiments each performed in triplicate or quadruplicate in ( a , b , d , e ). a , n = 4; b , n = 3; ( d , e ), CGNs, n = 3; HEK293 cells, n = 4. Data are analysed using one-way ANOVA with a Dunnett’s post-hoc multiple comparison test to determine significance (compared with brimonidine in a , b ). Data are analysed using unpaired t -test (two-tailed) in ( e ). **** p < 0.0001, *** p < 0.001, ** p < 0.01 and not significant (ns). The raw data and p -values are available in source data provided as a Source Data file.

Article Snippet: The blots were incubated with the primary monoclonal antibodies anti-GB1 mAb (1:1000, ab55051, Abcam, Shanghai, China), anti-Gβ 2 rabbit (1:1000, A9643, ABclonal Technology, Wuhan, China), anti-β-actin (1:3000, KM9001T, Sungene Biotech., Tianjin, China), the rabbit polyclonal antibodies anti-CB1 (1:1000, A1447, ABclonal, Wuhan, China), anti-α 2A AR (1:1000, A2809, ABclonal, Wuhan, China), anti-Gβ 1 (1:1000, A1867, ABclonal, Wuhan, China), anti-Gβ 3 (1:1000, A1387, ABclonal, Wuhan, China) and anti-Gβ 5 (1:1000, A4447, ABclonal, Wuhan, China).

Techniques: Transfection, Western Blot, Expressing, Comparison, Two Tailed Test