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

Journal: Frontiers in Neuroscience

Article Title: Role of Spinal Cord α 2 -Adrenoreceptors in Noradrenergic Inhibition of Nociceptive Transmission During Chemotherapy-Induced Peripheral Neuropathy

doi: 10.3389/fnins.2019.01413

Figure Lengend Snippet: Expression of α 2 A -AR in the spinal dorsal horn (30 days after the first paclitaxel injection). Representative photomicrographs of a α 2 A -AR positive pixels at laminae I–II are shown in (A,B) . The immunolabelling occurs in structures with neuronal morphology, which are better represented by arrows in (B) . Scale bar in (A,B) : 100 and 50 μm, respectively. (C,D) Show the size and the number of α 2 A -AR positive neurons, respectively, determined in laminae I-II in DMSO- and paclitaxel-injected animals. (E) Represents the percentage of α 2 A -AR positive pixels in laminae I-II of DMSO- and paclitaxel-injected animals. Data are presented as mean ± SD (DMSO n = 5; paclitaxel n = 5). (F) Depicts the quantification of α 2 A –AR assessed by western-blotting from DMSO- and paclitaxel-injected animals. GAPDH was used as a control of the western-blotting procedure. Data are presented as mean ± SD (DMSO n = 5; paclitaxel n = 5).

Article Snippet: For the α 2 A -AR immunoreaction, one in every fourth spinal sections was incubated with a rabbit-raised anti-α 2 A primary antibody (Neuromics; Cat. No. RA14110), diluted at 1:500, followed by incubation for 1 h with a donkey anti-rabbit Alexa 488 (Molecular Probes ® ; 1:1000).

Techniques: Expressing, Injection, Western Blot

Journal: The Journal of Veterinary Medical Science

Article Title: Inhibition of transient receptor potential vanilloid type 1 through α 2 adrenergic receptors at peripheral nerve terminals relieves pain

doi: 10.1292/jvms.21-0429

Figure Lengend Snippet: Sequences of primers for RT-PCR

Article Snippet: Subsequently, the sections were incubated with a guinea pig anti-TRPV1 antibody (Merck Millipore corporation, Temecula, CA, USA, Cat# AB5566, Lot# 3135959) diluted to 1:50, a rabbit anti-α 2A antibody (Cusabio Technology, Houston, TX, USA, Cat# CSB-PA007424, Lot# G1229Y) diluted to 1:100 or a rabbit anti-α 2C antibody (Cusabio Technology, Cat# CSB-PA000916, Lot# G1229Y) diluted to 1:100 overnight at 4°C.

Techniques:

Journal: The Journal of Veterinary Medical Science

Article Title: Inhibition of transient receptor potential vanilloid type 1 through α 2 adrenergic receptors at peripheral nerve terminals relieves pain

doi: 10.1292/jvms.21-0429

Figure Lengend Snippet: Subtypes of the α 2 adrenergic receptors detected in dorsal root ganglia. A. The real-time PCR amplification curves are shown in the left panel. A threshold was set for 3, and the Ct values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and α 2 receptors were obtained. The ΔCt values of α 2A (n=8), α 2B (n=8) and α 2C (n=8) receptors against GAPDH were calculated and are shown in the right panel. The columns represent the mean ± the standard error of the mean. B, C. Images of the immunoreactivities of TRPV1, α 2A and α 2C receptors in rat dorsal root ganglia. The TRPV1-, α 2A receptor- and α 2C receptor-like immunoreactivities were visualized by 3,3′-diaminobenzidine. Arrows, positively stained small-sized neurons; arrowheads, positively stained medium-sized neurons.

Article Snippet: Subsequently, the sections were incubated with a guinea pig anti-TRPV1 antibody (Merck Millipore corporation, Temecula, CA, USA, Cat# AB5566, Lot# 3135959) diluted to 1:50, a rabbit anti-α 2A antibody (Cusabio Technology, Houston, TX, USA, Cat# CSB-PA007424, Lot# G1229Y) diluted to 1:100 or a rabbit anti-α 2C antibody (Cusabio Technology, Cat# CSB-PA000916, Lot# G1229Y) diluted to 1:100 overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Amplification, Staining

Journal: The Journal of Neuroscience

Article Title: Differential Distribution of α 2A and α 2C Adrenergic Receptor Immunoreactivity in the Rat Spinal Cord

doi: 10.1523/JNEUROSCI.18-15-05928.1998

Figure Lengend Snippet: Characterization of α2A-AR-IR fibers in rat spinal cord. Single sections of lumbar spinal cord were double-labeled with α2A-AR antisera in combination with various other antisera. The first column(A, E, I,M, Q, U) depicts α2A-AR-IR (red). The second column (B, F, J,N, R, V) depicts immunoreactivity obtained with other antisera used in double-labeling (green). The third column(C, G, K,O, S, W) depicts digitally merged images of the first two columns. In merged images, the appearance of yellow may indicate colocalization, although higher magnification is required to distinguish colocalization from close apposition. The fourth column(D, H, L,P, T, X) depicts merged images obtained at higher magnification. Row 1(A–D) demonstrates that α2A-AR -IR and α2C-AR-IR are rarely colocalized. Row 2(E–H) demonstrates that α2A-AR-IR and SP-IR are highly colocalized. Row 3 (I–L) demonstrates that α2A-AR-IR and CGRP-IR fibers are highly, but not exclusively, colocalized. Row 4(M–P) demonstrates that α2A-AR-IR and ENK-IR are rarely colocalized. Row 5 (Q–T) demonstrates that α2A-AR-IR and DβH-IR are closely apposed, but not colocalized. Similarly, row 6(U–X) demonstrates that instances of colocalization between α2A-AR-IR and TH-IR could not be detected. SP, Substance P; CGRP, calcitonin gene-related peptide; ENK, leuenkephalin; DβH, dopamine β-hydroxylase; TH, tyrosine hydroxylase.

Article Snippet: Primary antisera were used at the following dilutions: rabbit anti-α 2A -AR, 1:1000; rabbit anti-α 2C -AR, 1:1000; guinea pig anti-α 2C -AR, 1:500; mouse anti-calcitonin gene-related peptide (CGRP), 1:2000 (Research Biochemicals, Natick, MA); mouse anti-dopamine-β-hydroxylase, 1:500 (Chemicon, Temecula, CA); rat anti-substance P (SP), 1:1000 (Research Biochemicals); rabbit anti-neurokinin-1 receptor (NK-1R) 1:500 ( Vigna et al., 1994 ; a gift from Dr. P. Mantyh, University of Minnesota, Minneapolis, MN); rabbit anti-somatostatin, 1:1000 ( Seybold and Elde, 1980 ); mouse anti-leuenkephalin, 1:500 (Sera-Lab, Sussex, UK); mouse anti-tyrosine hydroxylase, 1:500 (IncStar, Stillwater, MN); guinea pig anti-preprodynorphin, 1:500 ( Arvidsson et al., 1995b ); and mouse anti-GFAP, 1:500 (Sigma).

Techniques: Labeling

Journal: The Journal of Neuroscience

Article Title: Differential Distribution of α 2A and α 2C Adrenergic Receptor Immunoreactivity in the Rat Spinal Cord

doi: 10.1523/JNEUROSCI.18-15-05928.1998

Figure Lengend Snippet: Characterization of α2C-AR-IR fibers in rat spinal cord. Single sections of lumbar spinal cord were double-labeled with α2C-AR antisera in combination with various other antisera. The first column(A, E, I,M, Q, U,Y) depicts α2C-AR-IR (red). The second column(B, F, J,N, R, V, Z) depicts immunoreactivity obtained with other antisera used in double-labeling (green). The third column (C, G, K,O, S, W,AA) depicts digitally merged images of the first two columns. In merged images, the appearance of yellow may indicate colocalization, although higher magnification is required to distinguish colocalization from close apposition. The fourth column (D, H, L,P, T, X,BB) depicts merged images obtained at higher magnification. Row 1(A–D) demonstrates that α2C-AR-IR and SP-IR are rarely colocalized. Row 2 (E–H) demonstrates that α2C-AR-IR and CGRP-IR fibers are moderately colocalized.Row 3 (I–L) demonstrates that α2C-AR-IR and somatostatin-IR fibers are moderately colocalized. Row 4 (M–P) demonstrates that α2C-AR-IR and ENK-IR are moderately colocalized. Row 5(Q–T) demonstrates that α2C-AR-IR and NK-1R are not colocalized. Row 6 (U–X) demonstrates that α2C-AR-IR and GFAP-IR are very rarely colocalized.Row 7 (Y–BB) demonstrates that α2C-AR-IR and DβH-IR are not colocalized. A similar lack of colocalization was observed between α2C-AR-IR and TH-IR (data not shown). SP, Substance P; CGRP, calcitonin gene-related peptide; SOM, somatostatin; ENK, leuenkephalin; NK-1R, NK-1 receptor; GFAP, glial fibrillary acidic protein; DβH, dopamine β-hydroxylase.

Article Snippet: Primary antisera were used at the following dilutions: rabbit anti-α 2A -AR, 1:1000; rabbit anti-α 2C -AR, 1:1000; guinea pig anti-α 2C -AR, 1:500; mouse anti-calcitonin gene-related peptide (CGRP), 1:2000 (Research Biochemicals, Natick, MA); mouse anti-dopamine-β-hydroxylase, 1:500 (Chemicon, Temecula, CA); rat anti-substance P (SP), 1:1000 (Research Biochemicals); rabbit anti-neurokinin-1 receptor (NK-1R) 1:500 ( Vigna et al., 1994 ; a gift from Dr. P. Mantyh, University of Minnesota, Minneapolis, MN); rabbit anti-somatostatin, 1:1000 ( Seybold and Elde, 1980 ); mouse anti-leuenkephalin, 1:500 (Sera-Lab, Sussex, UK); mouse anti-tyrosine hydroxylase, 1:500 (IncStar, Stillwater, MN); guinea pig anti-preprodynorphin, 1:500 ( Arvidsson et al., 1995b ); and mouse anti-GFAP, 1:500 (Sigma).

Techniques: Labeling

Journal: The Journal of Veterinary Medical Science

Article Title: Inhibition of transient receptor potential vanilloid type 1 through α 2 adrenergic receptors at peripheral nerve terminals relieves pain

doi: 10.1292/jvms.21-0429

Figure Lengend Snippet: Sequences of primers for RT-PCR

Article Snippet: Subsequently, the sections were incubated with a guinea pig anti-TRPV1 antibody (Merck Millipore corporation, Temecula, CA, USA, Cat# AB5566, Lot# 3135959) diluted to 1:50, a rabbit anti-α 2A antibody (Cusabio Technology, Houston, TX, USA, Cat# CSB-PA007424, Lot# G1229Y) diluted to 1:100 or a rabbit anti-α 2C antibody (Cusabio Technology, Cat# CSB-PA000916, Lot# G1229Y) diluted to 1:100 overnight at 4°C.

Techniques:

Journal: The Journal of Veterinary Medical Science

Article Title: Inhibition of transient receptor potential vanilloid type 1 through α 2 adrenergic receptors at peripheral nerve terminals relieves pain

doi: 10.1292/jvms.21-0429

Figure Lengend Snippet: Subtypes of the α 2 adrenergic receptors detected in dorsal root ganglia. A. The real-time PCR amplification curves are shown in the left panel. A threshold was set for 3, and the Ct values of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and α 2 receptors were obtained. The ΔCt values of α 2A (n=8), α 2B (n=8) and α 2C (n=8) receptors against GAPDH were calculated and are shown in the right panel. The columns represent the mean ± the standard error of the mean. B, C. Images of the immunoreactivities of TRPV1, α 2A and α 2C receptors in rat dorsal root ganglia. The TRPV1-, α 2A receptor- and α 2C receptor-like immunoreactivities were visualized by 3,3′-diaminobenzidine. Arrows, positively stained small-sized neurons; arrowheads, positively stained medium-sized neurons.

Article Snippet: Subsequently, the sections were incubated with a guinea pig anti-TRPV1 antibody (Merck Millipore corporation, Temecula, CA, USA, Cat# AB5566, Lot# 3135959) diluted to 1:50, a rabbit anti-α 2A antibody (Cusabio Technology, Houston, TX, USA, Cat# CSB-PA007424, Lot# G1229Y) diluted to 1:100 or a rabbit anti-α 2C antibody (Cusabio Technology, Cat# CSB-PA000916, Lot# G1229Y) diluted to 1:100 overnight at 4°C.

Techniques: Real-time Polymerase Chain Reaction, Amplification, Staining

Journal:

Article Title: Aging-related hyperinflammation in endotoxemia is mediated by the ? 2A -adrenoceptor and CD14/TLR4 pathways

doi: 10.1016/j.lfs.2010.03.009

Figure Lengend Snippet: Proteins were extracted from splenic tissues of sham and LPS exposed young and aged rats and examined for CD14 (A) and TLR4 (B) protein by Western blotting. β-actin antibody was used as internal control for protein loading. Data are shown as CD14/β-actin ratio. Data are presented as mean ± SE (n=6) and compared by two-way ANOVA and Student-Newman-Keuls method: * p<0.05 versus respective sham; #p <0.05 versus young sham or LPS.

Article Snippet: The blots were incubated at 4°C overnight with either 1:300 dilution of anti-α 2A -AR, 1:500 dilution of anti-PDE4D, 1:1000 dilution of each of anti-CD14 or anti-TLR4 polyclonal antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Western Blot, Control