anti α 2a ar  (Proteintech)

Journal: Science Advances

Article Title: Structural insights into ligand recognition, activation, and signaling of the α 2A adrenergic receptor

doi: 10.1126/sciadv.abj5347

Figure Lengend Snippet: Cryo-EM density maps and models of the α 2A AR-GoA complex bound to Norepi ( A ), BRI ( B ), DEX ( C ), and OXY ( D ). The densities of the agonists (shown as sticks) are depicted as gray meshes. The maps are colored according to different subunits.

Article Snippet: The α 2A AR-GoA complex was then treated with 50 U of apyrase (NEB) on ice overnight and exchanged on an anti-Flag M1 column into a buffer containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.0075% lauryl maltose neopentyl glycol (MNG, NG310 Anatrace), 0.0025% GDN (GDN101, Anatrace), 0.001% CHS, 100 μM agonist, and 2 mM CaCl 2 in a stepwise manner.

Techniques: Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Structural insights into ligand recognition, activation, and signaling of the α 2A adrenergic receptor

doi: 10.1126/sciadv.abj5347

Figure Lengend Snippet: ( A ) Alignment of four structures of α 2A AR signaling complexes. ( B ) Superposition of Norepi, BRI, DEX, and OXY from cryo-EM structures. The imidazole moiety is highlighted by the dashed circle. ( C to F ) Detailed interactions of Norepi (C), BRI (D), DEX (E), and OXY (F) with α 2A AR. Residues within 4 Å of agonist are shown in sticks. The polar interactions are indicated by black dashed lines. ( G ) Superposition of the α 2A AR orthosteric binding pocket residues bound to four different agonists. ( H and I ) Concentration-response curves of different agonists for G protein activation (H) and β-arrestin-2 recruitment (I) for wild-type (wt) α 2A AR and receptor mutants D128 3.32 A, Y431 7.43 A, S215 5.42 A, and Y409 6.55 A, respectively. Except for the activation of D128 3.32 A, which is normalized to DEX for G protein activation and relative to basal for arrestin recruitment, receptor activation is shown relative to the maximum effect of Norepi. Data are presented as means ± SEM of 3 to 11 independent experiments with repeats in duplicate.

Article Snippet: The α 2A AR-GoA complex was then treated with 50 U of apyrase (NEB) on ice overnight and exchanged on an anti-Flag M1 column into a buffer containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.0075% lauryl maltose neopentyl glycol (MNG, NG310 Anatrace), 0.0025% GDN (GDN101, Anatrace), 0.001% CHS, 100 μM agonist, and 2 mM CaCl 2 in a stepwise manner.

Techniques: Cryo-EM Sample Prep, Binding Assay, Concentration Assay, Activation Assay

Journal: Science Advances

Article Title: Structural insights into ligand recognition, activation, and signaling of the α 2A adrenergic receptor

doi: 10.1126/sciadv.abj5347

Figure Lengend Snippet: ( A ) Detailed interactions of Norepi with β 1 AR [Protein Data Bank (PDB) code: 7BU6]. Residues within 4 Å of agonist are shown in sticks. The polar interactions are indicated by black dashed lines. ( B ) Superposition of orthosteric pockets of β 1 AR-Norepi and β 2 AR-Epi (epinephrine) (PDB code: 4LDO). ( C ) Detailed interactions of dopamine with D1R (PDB code: 7CKZ). The polar interactions are indicated by black dashed lines. ( D ) Superposition of orthosteric pockets of β 1 AR-Norepi and α 2A AR-Norepi. Residues within 4 Å of agonist are shown in sticks.

Article Snippet: The α 2A AR-GoA complex was then treated with 50 U of apyrase (NEB) on ice overnight and exchanged on an anti-Flag M1 column into a buffer containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.0075% lauryl maltose neopentyl glycol (MNG, NG310 Anatrace), 0.0025% GDN (GDN101, Anatrace), 0.001% CHS, 100 μM agonist, and 2 mM CaCl 2 in a stepwise manner.

Techniques:

Journal: Science Advances

Article Title: Structural insights into ligand recognition, activation, and signaling of the α 2A adrenergic receptor

doi: 10.1126/sciadv.abj5347

Figure Lengend Snippet: ( A ) Structural comparison of inactive α 2A AR bound to RS79948 and active α 2A AR bound to Norepi, with changes highlighted as red arrows. The distance is calculated between positions of Cα of residue 6.29 in TM6. ( B ) Conformational changes within the orthosteric pocket are shown from the extracellular side, with changes highlighted as red arrows. ( C ) Cross sections of α 2A AR bound to antagonist and agonist are shown, with the interior in black and the exosite highlighted. ( D ) Concentration-response curves of F427 7.39 mutant for different agonists toward G protein activation and β-arrestin-2 recruitment. Data are presented as means ± SEM of 4 to 10 independent experiments with repeats in duplicate.

Article Snippet: The α 2A AR-GoA complex was then treated with 50 U of apyrase (NEB) on ice overnight and exchanged on an anti-Flag M1 column into a buffer containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.0075% lauryl maltose neopentyl glycol (MNG, NG310 Anatrace), 0.0025% GDN (GDN101, Anatrace), 0.001% CHS, 100 μM agonist, and 2 mM CaCl 2 in a stepwise manner.

Techniques: Concentration Assay, Mutagenesis, Activation Assay

Journal: Science Advances

Article Title: Structural insights into ligand recognition, activation, and signaling of the α 2A adrenergic receptor

doi: 10.1126/sciadv.abj5347

Figure Lengend Snippet: ( A and B ) Superposition of the G protein coupling interfaces of α 2A AR-GoA, α 2B AR-GoA, and β 2 AR-Gs complexes, using receptor for alignment. ( C to H ) Detailed interactions of α 2A AR with Goα (C and D), α 2B AR with Goα (E and F), and β 2 AR with Gsα (G and H). The polar interactions are indicated by red dashed lines.

Article Snippet: The α 2A AR-GoA complex was then treated with 50 U of apyrase (NEB) on ice overnight and exchanged on an anti-Flag M1 column into a buffer containing 20 mM Hepes (pH 7.5), 100 mM NaCl, 0.0075% lauryl maltose neopentyl glycol (MNG, NG310 Anatrace), 0.0025% GDN (GDN101, Anatrace), 0.001% CHS, 100 μM agonist, and 2 mM CaCl 2 in a stepwise manner.

Techniques:

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

Journal: Frontiers in Immunology

Article Title: Sympathetic Denervation Ameliorates Renal Fibrosis via Inhibition of Cellular Senescence

doi: 10.3389/fimmu.2021.823935

Figure Lengend Snippet: The α 2A -adrenergic receptor is responsible for NE-induced tubular epithelial cell senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative images of immunohistochemistry for α 2A -adrenergic receptors (α 2A -AR) in sham-operated, UUO/UIRI, mice and DNx+UUO/UIRI mice. Scale bar, 20μm. (C, D) After starving for 8 hours, TKPTS cells were cultured with PBS or NE (10 nM) for 48 hours. α 2A -AR expressions were tested by immunofluorescence (C) (Scale bar, 20 μm) and Western Blot (D) (n=3 in each group). (E, F) After pretreatment with PBS or α 2A -adrenergic antagonist (BRL4408, BRL, 10 μM) for 1 hour, TKPTS cells were incubated with NE (10 nM) for 48 hours. (E) Representative Western blotting of p53, p21, and γ-H2AX in each group with densitometry analysis (n=4-5). (F) mRNA expressions of p53, p21, p16, HMGA2, H2AX, TGF-β1, IL-6, IL-8, and IL-1β were analyzed using qRT-PCR analysis (n=3–8 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represented means ± SEM.

Article Snippet: Western blot analyses were conducted following the manufacturer’s instructions with specific primary antibodies: fibronectin (15613-1-AP, Proteintech, Rosemont, IL, USA), College 1 (14695-1-AP, Proteintech), α-SMA (ab5694, Abcam), tyrosine hydroxylase (TH, ab112, Abcam), p53 (A3185, ABclonal Technology, China), p21 (14-6715-81, Invitrogen), γ-H2AX (#9718, Cell Signaling Technology, Danvers, MA, USA), α 2A -AR (DF3076, Affinity Biosciences), β-arrestin2 (10171-1-AP, Proteintech), VDAC1 (55259-1-AP, Proteintech), COX IV (11242-1-AP, Proteintech), and NF-κB p65 (sc-8008, Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Cell Culture, Immunofluorescence, Western Blot, Incubation, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Sympathetic Denervation Ameliorates Renal Fibrosis via Inhibition of Cellular Senescence

doi: 10.3389/fimmu.2021.823935

Figure Lengend Snippet: β-arrestin2 is a target of α 2A -AR and knockdown of β-arrestin2 ameliorates NE-induced tubular cellular senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative images of immunohistochemistry for β-arrestin2 in sham-operated, UUO/UIRI mice, and DNx+UUO/UIRI mice. Scale bar, 20 μm. (C, D) Representative Western blotting of β-arrestin2 (β-arr2) in sham-operated, UUO/UIRI, and DNx+UUO/UIRI kidneys with densitometry analysis (n=3–6 in each group). (E–G) TKPTS cells were cultured with PBS or NE (10 nM) for 48 hours. (E) Representative images of immunofluorescence for β-arrestin2 in each group. Scale bar, 20μm. (F) Cells were double-labeled with α 2A -AR (green) and β-arrestin2 (red), and cell nuclei were enhanced by staining with DAPI (blue). Colocalization of α 2A -AR (green), and β-arrestin2 (red) was visualized as yellow in merged images. Scale bar, 20μm. (G) Interactions between β-arrestin2 and α 2A -AR were examined by immunoprecipitation (IP) with an anti-β-arrestin2 antibody, followed by immunoblotting (IB) with anti-α 2A -AR antibodies. (H, I) After an hour of pretreatment with PBS or BRL4408, TKPTS cells were stimulated with NE (10 nM) for 48 hours. (H) Representative Western blotting of β-arrestin2 (β-arr2) in TKPTS cells treated with PBS, NE, and NE+BRL (100 nM, 1 μM, 10 μM, and 100 μM) (n=3 in each group). (I) mRNA expression of β-arrestin2 in PBS, NE, and NE+BRL (10 μM)-treated TKPTS cells as measured by qRT-PCR analysis (n=3–6 in each group). (J, K) TKPTS cells were transfected with scrambled siRNA or three β-arrestin2 siRNAs (si-Arrb2-1, si-Arrb2-2, or si-Arrb2-3) for 48 hours. β-arrestin2 (β-arr2) expression was examined by Western blot analysis (J) and qRT-PCR analysis (K) (n=3 in each group). (L, M) TKPTS cells were transfected with scrambled siRNA (si-control) or β-arrestin2 siRNA (si-Arrb2) 24 hours before NE (10 nM) treatment. Representative Western blotting of p53 and p21 in each group with densitometry analysis (L) (n=4–5 in each group). mRNA expressions of p21, H2AX, TGF-β1, IL-6, and IL-8 were tested by qRT-PCR analysis (M) (n=3–6 in each group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Bars represented means ± SEM.

Article Snippet: Western blot analyses were conducted following the manufacturer’s instructions with specific primary antibodies: fibronectin (15613-1-AP, Proteintech, Rosemont, IL, USA), College 1 (14695-1-AP, Proteintech), α-SMA (ab5694, Abcam), tyrosine hydroxylase (TH, ab112, Abcam), p53 (A3185, ABclonal Technology, China), p21 (14-6715-81, Invitrogen), γ-H2AX (#9718, Cell Signaling Technology, Danvers, MA, USA), α 2A -AR (DF3076, Affinity Biosciences), β-arrestin2 (10171-1-AP, Proteintech), VDAC1 (55259-1-AP, Proteintech), COX IV (11242-1-AP, Proteintech), and NF-κB p65 (sc-8008, Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Western Blot, Cell Culture, Immunofluorescence, Labeling, Staining, Immunoprecipitation, Expressing, Quantitative RT-PCR, Transfection

Journal: Frontiers in Immunology

Article Title: Sympathetic Denervation Ameliorates Renal Fibrosis via Inhibition of Cellular Senescence

doi: 10.3389/fimmu.2021.823935

Figure Lengend Snippet: α 2A -AR/β-arrestin2/NF-κB signaling contributes to mitochondrial dysfunction and cellular senescence. (A, B) Renal denervation (DNx) or sham operation was performed 2 days before UUO or UIRI in the left kidneys of mice. Representative Western blotting of VDAC1 and COX IV in each group with densitometry analysis (n=6). (C) Representative Western blotting of VDAC1 and COX IV in TKPTS cells treated with PBS or NE (10 nM) for 48 hours with densitometry analysis (n=3–6 in each group). (D–F) TKPTS cells were transfected with scrambled siRNA (si-control) or β-arrestin2 siRNA (si-Arrb2) for 24 hours and then incubated with NE (10 nM) for 48 hours. Mitochondrial ultrastructure was observed by transmission electron microscope (D) . Scale bar, 1 μm. Mitochondrial mass was tested by MitoTracker deep red probe (100nM) staining (E) . Scale bar, 50 μm. Mitochondrial membrane potential was examined by TMRE fluorescent dye (200 nM) (F) . Scale bar, 20 μm. (G) Representative Western blotting of NF-κB p65 in TKPTS cells incubated with PBS, NE, and NE+BRL (10 μM) with densitometry analysis (n=4–6 in each group). (H) Representative Western blotting of NF-κB p65 in TKPTS cells of PBS, NE, and NE+si-control, NE+si-Arrb2 with densitometry analysis (n=3-4 in each group). *P < 0.05, **P < 0.01, ***P < 0.001. Bars represented means ± SEM.

Article Snippet: Western blot analyses were conducted following the manufacturer’s instructions with specific primary antibodies: fibronectin (15613-1-AP, Proteintech, Rosemont, IL, USA), College 1 (14695-1-AP, Proteintech), α-SMA (ab5694, Abcam), tyrosine hydroxylase (TH, ab112, Abcam), p53 (A3185, ABclonal Technology, China), p21 (14-6715-81, Invitrogen), γ-H2AX (#9718, Cell Signaling Technology, Danvers, MA, USA), α 2A -AR (DF3076, Affinity Biosciences), β-arrestin2 (10171-1-AP, Proteintech), VDAC1 (55259-1-AP, Proteintech), COX IV (11242-1-AP, Proteintech), and NF-κB p65 (sc-8008, Santa Cruz Biotechnology).

Techniques: Western Blot, Transfection, Incubation, Transmission Assay, Microscopy, Staining, Membrane

Journal: Journal of Cellular and Molecular Medicine

Article Title: The α2AR/Caveolin‐1/p38MAPK/NF‐κB axis explains dexmedetomidine protection against lung injury following intestinal ischaemia‐reperfusion

doi: 10.1111/jcmm.16614

Figure Lengend Snippet: Dex reduces lung injury following intestinal I/R via Cav‐1‐dependent p38MAPK/NF‐κB pathway inactivation. A, Comparison of the down‐regulation for Cav‐1 protein using three anti‐Cav‐1 shRNA constructs. * P < .05 compared with the mock group. Pre‐injections were applied four days before intestinal I/R injury. B, RT‐qPCR of Cav‐1 mRNA expression and Western blot analysis of Cav‐1, p38, p‐p38, p‐p65 and p65 expression in rat lung tissues with Cav‐1 overexpression or knockdown before intestinal I/R injury. C, ELISA detection of TNF‐α expression in rat lung tissues with Cav‐1 overexpression or knockdown before intestinal I/R injury. D, ELISA detection of IL‐1β expression in rat lung tissues with Cav‐1 overexpression or knockdown before intestinal I/R injury. E, ELISA detection of IL‐6 expression in rat lung tissues with Cav‐1 overexpression or knockdown before intestinal I/R injury. F, Lung injury score of rat lung tissues with Cav‐1 overexpression or knockdown before intestinal I/R injury. G, Apoptosis rate of rat lung tissues with Cav‐1 overexpression or knockdown before intestinal I/R injury. * P < .05 vs sham group, # P < .05 vs I/R + oe‐NC group. H, Western blot analysis of Cav‐1, p38, p‐p38, p‐p65 and p65 in rat lung tissues upon Dex treatment and/or Cav‐1 knockdown prior to intestinal I/R injury. I, ELISA detection of TNF‐α expression in rat lung tissues upon Dex treatment and/or Cav‐1 knockdown prior to intestinal I/R injury. J, ELISA detection of IL‐1βexpression in rat lung tissues upon Dex treatment and/or Cav‐1 knockdown prior to intestinal I/R injury. K, ELISA detection of IL‐6 expression in rat lung tissues upon Dex treatment and/or Cav‐1 knockdown prior to intestinal I/R injury. L, Lung injury score of rat lung tissues upon Dex treatment and/or Cav‐1 knockdown prior to intestinal I/R injury. M, Apoptosis rate of rat lung tissues upon Dex treatment and/or Cav‐1 knockdown prior to intestinal I/R injury, detected by TUNEL staining. * P < .05 vs sham group, # P < .05 vs I/R + Dex +sh‐NC group. Data among multiple groups were analysed by one‐way ANOVA with Tukey's post hoc test

Article Snippet: The PVDF membrane was blocked in 5% skimmed milk at room temperature for 1 hours and incubated overnight at 4°C with the diluted primary antibodies: rabbit anti‐α 2A ‐AR (1:1000, 14266‐1‐AP, Proteintech ProteinTech Group), rabbit anti‐Cav‐1 (1:1000, ab32577, Abcam Inc.), rabbit anti‐p38 (1:1000, ab31828, Abcam), rabbit anti‐phosphorylated (p)‐p38 (1:1000, ab4822, Abcam), rabbit anti‐NF‐κBp65 (1:1000, ab16502, Abcam), rabbit anti‐NF‐κB‐p‐p65 (1:1000, ab86299, Abcam) and rabbit anti‐Actin (1:1000, ab179467, Abcam).

Techniques: Comparison, shRNA, Construct, Quantitative RT-PCR, Expressing, Western Blot, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Staining