anti a2bars rabbit polyclonal antibodies  (Santa Cruz Biotechnology)

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

doi:

Figure Lengend Snippet: Primers of the genes and the size of the products

Article Snippet: After blocking with 5% bovine serum albumin in TBS-T (50 mMTris–HCl [pH 7.5], 140 mM NaCl, and 0.1% Tween) for 2 h, the membranes were incubated overnight at 4°C with polyclonal rabbit antibodies against A2BAR (1:250,Santa Cruz), claudin-1 (1:500, Abcam) and ZO-1 (1:500, Santa Cruz), monoclonal anti-occludin (1:250, Santa Cruz) and anti-GAPDH (1:1000, Goodhere Biotechnology, Hangzhou, China).

Techniques:

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

doi:

Figure Lengend Snippet: Changes in A2BAR expression under hypoxia and intestinal I/R models. A. Caco-2 cells were exposed to normoxia or hypoxia for indicated time. Total RNA was isolated, and A2BAR mRNA levels were determined by RT-PCR. Data were calculated relative to β-actin and expressed as fold change relative to normoxia. B. Induction of intestinal epithelia A2BAR protein by hypoxia. Shown here is a representative Western blot of A2BAR following incubation for indicated periods of hypoxia, with β-actin as a control. C. Induction of intestinal epithelial A2BAR mRNA levels by IR. Results are derived from three experiments (*: Different from normoxia and Sham group, P < 0.01). F-H. Immunohistochemistry for A2BAR. A2BAR expression was detected in I/R 6 h, compared with the Sham group. F. Negative control; G. Sham group exhibit weak staining; H) The structures of intestinal villus were significantly damaged of I/R injury when compared with the sham group (n = 6 per group). I. Induction of A2BAR in Caco-2 cell lines in normoxic or hypoxic conditions. Immunocytofluorescence labeling of A2BAR (red) in Caco-2 cells cultured for 6 h under normoxia, hypoxia and hypoxia plus PSB1115 (10 μM) is shown. Cellular nuclei are labeled with DAPI (blue). The positive fluorescence of A2BAR (red) is higher under the hypoxic condition than under the normoxic condition.

Article Snippet: After blocking with 5% bovine serum albumin in TBS-T (50 mMTris–HCl [pH 7.5], 140 mM NaCl, and 0.1% Tween) for 2 h, the membranes were incubated overnight at 4°C with polyclonal rabbit antibodies against A2BAR (1:250,Santa Cruz), claudin-1 (1:500, Abcam) and ZO-1 (1:500, Santa Cruz), monoclonal anti-occludin (1:250, Santa Cruz) and anti-GAPDH (1:1000, Goodhere Biotechnology, Hangzhou, China).

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation, Derivative Assay, Immunohistochemistry, Negative Control, Staining, Labeling, Cell Culture, Fluorescence

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

doi:

Figure Lengend Snippet: Role of an A2BAR antagonist on TJ mRNA levels under hypoxia and I/R. Caco-2 cells were pretreated with or without PSB1115, and the monolayers were then subjected to hypoxia for 6 h. The mRNA levels of the TJs were determined using RT-PCR. The data are the mean ± SD (n = 6). A-C. Hypoxia down-regulated claudin-1, occludin and ZO-1 mRNA expression, and PSB1115 attenuated the down-regulation of these TJs as induced by hypoxia. D-F. I/R down-regulated claudin-1, occludin and ZO-1 mRNA expression, and PSB1115 attenuated the down-regulation of TJ mRNA expression induced by I/R (*P < 0.05, **P < 0.01 compared to the Nx group and Sham group; ***P < 0.05, compared to the Hx. #P < 0.01, compared to the I/R group).

Article Snippet: After blocking with 5% bovine serum albumin in TBS-T (50 mMTris–HCl [pH 7.5], 140 mM NaCl, and 0.1% Tween) for 2 h, the membranes were incubated overnight at 4°C with polyclonal rabbit antibodies against A2BAR (1:250,Santa Cruz), claudin-1 (1:500, Abcam) and ZO-1 (1:500, Santa Cruz), monoclonal anti-occludin (1:250, Santa Cruz) and anti-GAPDH (1:1000, Goodhere Biotechnology, Hangzhou, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

doi:

Figure Lengend Snippet: Effect of an A2BAR antagonist on the abundance of TJ proteins in Caco-2 cells exposed to hypoxia and I/R intestinal tissue. A-D. Caco-2 cells were cultured in MEM for 7 days under normoxia, incubated for 24 h in serum-free medium, and then exposed to hypoxia in the absence or presence of PSB1115 (10 μM) for 6 h. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies against claudin-1, occludin and ZO-1. The amount of each TJ protein was normalized to the GAPDH and then expressed relative to the corresponding normalized value for the normoxic condition. E-H. Claudin-1, occludin and ZO-1 was detected in Sham, I/R and I/R+PSB1115 groups. The amount of each TJ protein was normalized to the GAPDH and then expressed relative to the corresponding normalized value for the Sham group. The data are the means ± SD from three independent experiments. (*P < 0.05, **P < 0.01, compared to the Nx group and Sham group; ***P < 0.05, compared to the Hx group and IR group).

Article Snippet: After blocking with 5% bovine serum albumin in TBS-T (50 mMTris–HCl [pH 7.5], 140 mM NaCl, and 0.1% Tween) for 2 h, the membranes were incubated overnight at 4°C with polyclonal rabbit antibodies against A2BAR (1:250,Santa Cruz), claudin-1 (1:500, Abcam) and ZO-1 (1:500, Santa Cruz), monoclonal anti-occludin (1:250, Santa Cruz) and anti-GAPDH (1:1000, Goodhere Biotechnology, Hangzhou, China).

Techniques: Cell Culture, Incubation, Western Blot

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

doi:

Figure Lengend Snippet: A2BAR inhibition prevents the disruption of TJs induced by hypoxia. Caco-2 cells were cultured in MEM for 7 days under the normoxic condition, incubated for 24 h in serum-free medium, then exposed to hypoxia in the absence or presence of PSB1115 (10 μM) and then exposed to hypoxia for 6 h. The cells were subjected to an immunofluorescence analysis using antibodies against claudin-1 (A), occludin (B) and ZO-1 (C). The data are representative of three independent experiments.

Article Snippet: After blocking with 5% bovine serum albumin in TBS-T (50 mMTris–HCl [pH 7.5], 140 mM NaCl, and 0.1% Tween) for 2 h, the membranes were incubated overnight at 4°C with polyclonal rabbit antibodies against A2BAR (1:250,Santa Cruz), claudin-1 (1:500, Abcam) and ZO-1 (1:500, Santa Cruz), monoclonal anti-occludin (1:250, Santa Cruz) and anti-GAPDH (1:1000, Goodhere Biotechnology, Hangzhou, China).

Techniques: Inhibition, Cell Culture, Incubation, Immunofluorescence

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions

doi:

Figure Lengend Snippet: Effect of an A2BAR antagonist on the hypoxia-induced the changes in the TER in Caco-2 monolayers and I/R-induced changes in TER in intestinal epithelia. Hypoxia-induced reduction in the TER in Caco-2 monolayers and IR-induced reduction the TER in intestinal epithelia can be prevented by A2BAR antagonist pretreatment. The results are expressed as the mean ± SD (n = 6). A. **P < 0.01, compared to the Nx group; ***P < 0.05, compared to the Hx group. B. **P < 0.01, compared to the Sham group; #P < 0.01, compared to I/R group.

Article Snippet: After blocking with 5% bovine serum albumin in TBS-T (50 mMTris–HCl [pH 7.5], 140 mM NaCl, and 0.1% Tween) for 2 h, the membranes were incubated overnight at 4°C with polyclonal rabbit antibodies against A2BAR (1:250,Santa Cruz), claudin-1 (1:500, Abcam) and ZO-1 (1:500, Santa Cruz), monoclonal anti-occludin (1:250, Santa Cruz) and anti-GAPDH (1:1000, Goodhere Biotechnology, Hangzhou, China).

Techniques:

Journal:

Article Title: Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

doi: 10.4049/jimmunol.0903035

Figure Lengend Snippet: (A-D): Pulmonary epithelial cells (A549) or vascular endothelia (HMEC-1) were exposed to indicated concentrations of inflammatory mediators, and A2BAR transcript levels were determined by real-time RT-PCR following 0–24h of exposure time. Data were calculated relative to the internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (0h of exposure) ± SD at each indicated time (n=4). (E) Degradation curve of A2BAR mRNA in HMEC-1 cells treated with PGE2 and challenged with DRB (n=3; *p<0.005 by ANOVA). (F). A2BAR expression in primary pulmonary endothelial cells treated with IL-6 (HMVEC-L) (n=4). (G) Western-blot analysis of A2BAR protein following 24h of IL-6 stimulation at indicated concentrations. The same blot was stripped and re-probed for murine β-actin to control for loading conditions. One representative of three Western blots is displayed.

Article Snippet: Samples were incubated for 60 minutes with the following antibodies: Polyclonal rabbit anti-A2BAR antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1000 as primary antibody, rabbit Ig Fraction (Dako Cytomation, Glostrup, Denmark) as negative control.

Techniques: Quantitative RT-PCR, Control, Expressing, Western Blot

Journal:

Article Title: Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

doi: 10.4049/jimmunol.0903035

Figure Lengend Snippet: Mice were exposed to 30 min of LPS inhalation, and animals were sacrificed after 4h. (A) Pulmonary A2BAR transcript levels were assessed by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (-LPS) ± SD (n=9). Western-blot analysis of A2BAR protein following in vivo exposure to inhaled LPS. The same blot was stripped and re-probed for murine β-actin to control for loading conditions. One representative of four Western blots is displayed. (C) Pulmonary immunohistochemistry for the A2BAR following LPS exposure. Lungs from mice exposed to 30 min of LPS inhalation or vehicle control (Vehicle) were harvested. Sections were stained with antibodies specifical for murine A2BAR (green), or isotype controls. 4′,6-diamidino-2-phenylindole was used for nuclear counterstain (blue) (magnification, 400×). One representative image from 3 pulmonic sections is displayed.

Article Snippet: Samples were incubated for 60 minutes with the following antibodies: Polyclonal rabbit anti-A2BAR antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1000 as primary antibody, rabbit Ig Fraction (Dako Cytomation, Glostrup, Denmark) as negative control.

Techniques: Quantitative RT-PCR, Control, Western Blot, In Vivo, Immunohistochemistry, Staining

Journal:

Article Title: Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

doi: 10.4049/jimmunol.0903035

Figure Lengend Snippet: 8–12 week old C57BL/6J mice matched in age, gender and weight were pre-exposed to 30 min of inhaled A2BAR antagonist (MRS1754) or vehicle, followed by 30 min of LPS or vehicle inhalation. Animals were sacrificed after 30 min and pulmonary transcript levels of (A) IL-1β, (B) IL-6 or (C) TNF-α were assessed by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (−LPS, −MRS1754) ± SD (n=4–7). (D) Western-blot analysis of IL-6 protein assessed by Western blot. The same blot was stripped and re-probed for murine β-actin to control for loading conditions. One representative of three Western blots is displayed. Note enhanced lung inflammation following A2BAR antagonist treatment following LPS exposure.

Article Snippet: Samples were incubated for 60 minutes with the following antibodies: Polyclonal rabbit anti-A2BAR antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1000 as primary antibody, rabbit Ig Fraction (Dako Cytomation, Glostrup, Denmark) as negative control.

Techniques: Quantitative RT-PCR, Control, Western Blot

Journal:

Article Title: Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

doi: 10.4049/jimmunol.0903035

Figure Lengend Snippet: Endotoxin-induced acute lung injury was induced in 8–12 week old A2BAR−/− mice or littermate controls matched in age, gender and weight by inhalation of LPS over 30 min. Controls were treated with inhaled vehicle. Animals were sacrificed after 4h and pulmonary transcript levels of (A) IL-1β, (B) IL-6 or (C) TNF-α were assessed by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (-LPS) ± SD (n=5–13). (D) Western-blot analysis of IL-6 protein assessed by Western blot. The same blot was stripped and re-probed for murine β-actin to control for loading conditions. One representative of three Western blots is displayed. (E) Assessment of pulmonary edema by measurement of lung water content (n=5–6). (F) Measurement of pulmonary myeloperoxidase (MPO) as indicator for neutrophil accumulation (n=5–9). Note enhanced lung inflammation and pulmonary edema in A2BAR−/− mice following LPS exposure.

Article Snippet: Samples were incubated for 60 minutes with the following antibodies: Polyclonal rabbit anti-A2BAR antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1000 as primary antibody, rabbit Ig Fraction (Dako Cytomation, Glostrup, Denmark) as negative control.

Techniques: Quantitative RT-PCR, Control, Western Blot

Journal:

Article Title: Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

doi: 10.4049/jimmunol.0903035

Figure Lengend Snippet: Endotoxin-induced acute lung injury was induced in 8–12 week old A2BAR−/− mice or littermate controls matched in age, gender and weight by inhalation of LPS over 30 min. Controls were treated with inhaled vehicle. Animals were sacrificed, and pulmonary histology was assessed by a pathologist blinded to the treatment group. (A) Lung injury score (mean ± range, n=4). (B) Representative H&E staining (magnification 400x; one representative slide of 4). Note enhanced lung inflammation and tissue injury in A2BAR−/− mice exposed to LPS.

Article Snippet: Samples were incubated for 60 minutes with the following antibodies: Polyclonal rabbit anti-A2BAR antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1000 as primary antibody, rabbit Ig Fraction (Dako Cytomation, Glostrup, Denmark) as negative control.

Techniques: Staining

Journal:

Article Title: Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

doi: 10.4049/jimmunol.0903035

Figure Lengend Snippet: 8 to 12 week old A2BAR bone marrow chimeric mice (A2BAR+/+ → A2BAR+/+, WT → WT; A2BAR−/− → A2BAR+/+, WT → KO; A2BAR+/+ → A2BAR−/−, WT → KO and A2BAR−/− → A2BAR−/−, KO → KO) matched in age, gender and weight were exposed to 30 min of LPS or vehicle inhalation. Animals were sacrificed after 4h and (A) pulmonary transcript levels of IL-6 were determined by real-time RT-PCR. Data were calculated relative to internal housekeeping gene (β-actin) and are expressed as mean fold change compared with control (WT → WT treated with vehicle) ± SD (n=4–7).

Article Snippet: Samples were incubated for 60 minutes with the following antibodies: Polyclonal rabbit anti-A2BAR antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1000 as primary antibody, rabbit Ig Fraction (Dako Cytomation, Glostrup, Denmark) as negative control.

Techniques: Quantitative RT-PCR, Control

Journal:

Article Title: Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury

doi: 10.4049/jimmunol.0903035

Figure Lengend Snippet: (A-C) 8–12 week old C57BL/6J mice matched in age, gender and weight were pre-treated with A2BAR agonist (BAY 60-6583, 2mg/kg i.p.) or vehicle, followed by 30 min of LPS or vehicle inhalation. Animals were sacrificed after 30 min and pulmonary myeloperoixidase (A), IL-6 protein levels measured by ELISA (B) or lung water content (C) were assessed (n=4). (D-E) Similar studies in gene-targeted mice for the A2BAR (A2BAR−/− mice; n=4). Note attenuated lung inflammation and pulmonary edema with A2BAR agonist treatment only in wild-type mice.

Article Snippet: Samples were incubated for 60 minutes with the following antibodies: Polyclonal rabbit anti-A2BAR antibody (Santa Cruz Biotechnology, Inc.) at a dilution of 1:1000 as primary antibody, rabbit Ig Fraction (Dako Cytomation, Glostrup, Denmark) as negative control.

Techniques: Enzyme-linked Immunosorbent Assay