The PVN tissue was homogenized in a lysis buffer, and the protein concentration in the supernatant was measured with the BCA protein assay Kit (Pierce, Rockford, IL, USA). Equivalent amounts of protein were separated on 12% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). The membranes were blocked with 5% nonfat dry milk and then incubated using a primary antibody at 4°C overnight. The following primary antibodies used in this study have been verified in previous published literatures: anti-renin (sc-22752) [20 (link)], anti-AT1-R (sc-1173) [21 (link),22 (link)], anti-AT2-R (sc-9040) [23 (link)], anti-ACE-1 (sc-20791) [22 (link)], anti-ACE-2 (sc-20998) [24 (link)], anti-TNF-α (sc-1350) [25 (link)], anti-IL-1β (sc-7884) [25 (link)], anti-IL-10 (sc-57245) [26 (link)], and anti- β-actin (sc-47778) [22 (link)] (all antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA). After three washings, the membranes were incubated with horseradish peroxidase-conjugated second antibody (Santa Cruz Biotechnology Inc, Santa Cruz, CA) for 1 h at room temperature. The signal was visualized using the enhanced chemiluminescence (ECL) detection system (Amersham), and the densities of the immunobands were quantitated using NIH ImageJ software (Bethesda, MD, USA). All data were corrected by β-actin.
Anti at1r
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-AT1R is a laboratory reagent used in research applications. It functions as an antibody that specifically binds to the angiotensin II type 1 receptor (AT1R). This receptor is involved in the regulation of blood pressure and fluid balance in the body. The Anti-AT1R product is designed to facilitate the study of the AT1R and its associated signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Anti at2r, by Santa Cruz Biotechnology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti renin, by Santa Cruz Biotechnology (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated second antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Anti ace2, by Santa Cruz Biotechnology (1 mentions)
Anti tnf α, by Santa Cruz Biotechnology (1 mentions)
Anti il 1β, by Santa Cruz Biotechnology (1 mentions)
Anti il 10, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence ecl substrate, by Thermo Fisher Scientific (1 mentions)
Anti ace, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Anti ace2, by Abcam (1 mentions)
Nitrotyrosine, by Cayman Chemical (1 mentions)
Ang 2, by Novus Biologicals (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Metamorpho version 6, by Olympus (1 mentions)
Streptavidin biotin system, by Agilent Technologies (1 mentions)
9 protocols using anti at1r
1
Western Blot Analysis of Renin-Angiotensin System
2
Quantifying Myocardial Protein Expression
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Protein expression levels of ACE, ACE2, AT1R, and MasR in cardiac tissue were determined using western blotting as previously described [24 (link)]. In brief, frozen cardiac tissues were thawed, ground, and homogenized in the lysis buffer that contained protease inhibitors, surfactants, as well as phosphatase inhibitors. Protein samples were loaded in 8% or 10% gels for sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The resolved proteins were electrically transferred to the nitrocellulose membranes and detected using specific antibodies, including anti-ACE (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc-23,908; lot no. C1319), anti-ACE2 (Abcam, UK; cat no. ab108252; lot no. GR145000–28), anti-AT1R (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc515884; lot no. J0319), and anti-MasR (SantaCruz Biotechnology, Inc., CA, USA; cat no. sc-390,453; lot no. A1419), to quantify myocardial protein levels. The primary antibody-antigen complexes and the horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated and then visualized using an enhanced chemiluminescence (ECL) substrate (Thermo Fisher Scientific Inc., USA) and ECL hyperfilm (GE Healthcare Pvt. Ltd., UK). Blots were inspected by ImageJ software (NIH, USA). Results were normalized with the beta-actin (Sigma-Aldrich, USA; cat no. A5441) as the internal standard.
3
Western Blot Analysis of Oxidative Stress Markers
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Western blot analyses were conducted as previously described [4 (link)] using anti-AT1R (Santa Cruz, 1 : 1000), -AngII (Novus, 1 : 1000), -Nox2 (BD transduction, 1 : 5000), -Nox4 (Santa Cruz, 1 : 5000), -nitrotyrosine (Cayman, 1 : 1000), -Ly6G (ebioscience, 1 : 1000), and -GAPDH (Santa Cruz, 1 : 5000) antibodies. Densities of the blots were quantified using the ImageJ program (NIH, Bethesda, MD).
4
Quantitative Immunohistochemical Analysis of TGF-β and AT1R
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Immunohistochemistry was performed as described previously [14 (link)]. Paraffin-embedded tissues were cut into sections 4-µm thick, mounted on glass slides, and stained using indirect immunoperoxidase. The slides were processed for immunodetection of transforming growth factor β (TGF-β) and angiotensin II type 1 receptor (AT1R) with antibodies specific for TGF-β (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-AT1R (Santa Cruz Biotechnology) respectively. Diaminobenzidine (Sigma Chemical Co., St. Louis, MO, USA) was used as a chromogen. All samples were evaluated under an Olympus BX51 microscope. The size of the area stained positively for TGF-β (as a percentage of the total area in 10 separate fields of each section under ×200 magnification) was determined using a digital camera-based image analyzer (Metamorpho version 6.3, Olympus).
5
Adrenal Gland Immunohistochemistry Protocol
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The adrenal glands and left kidneys fixed with 4% paraformaldehyde were embedded in paraffin and sectioned (5 μm thickness). Sections had antigen retrieval performed by citrate buffer at pH 6.0 and incubated for 30 min at 60°C. Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide (H2O2), and nonspecific binding of the polyclonal antibodies was blocked by incubation 5% (w/v) BSA. Subsequently, sections were incubated with antibodies, and these reactions were amplified using a biotin–streptavidin system (Dako, USA). Immunoreactive products were visualized using diaminobenzidine (DAB) reagent (Dako, USA) and counter-stained with hematoxylin. We used anti-AT1R (sc-515884), antirenin (sc-137252), anti-PGC1-α (sc-518025), anticytochrome C (sc-13156), anti-Bax (sc-20067), anti-Bcl-2 (sc-7382), anti-EEA1 (sc-137130), and Rab 7 (sc-81922) (dilution 1 : 200, Santa Cruz Biotechnology, CA, USA, for all) antibodies. The immunostaining of the zona glomerulosa adrenal was observed under a light microscope equipped with a CCD camera (Olympus BX53 with the camera Olympus DP72, Japan).
6
Western Blot Analysis of Adrenergic Receptors in Rat Renal Cortex
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Total protein was extracted after homogenizing the rat renal cortex, and the concentration was determined with a bicinchoninic acid protein assay kit. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes, which were incubated with the primary antibodies rabbit anti-β1-AR (1:200), anti-β2-AR (1:200), anti-β3-AR (1:200), anti-α1A-AR (1:200), anti-α1B-AR (1:200), anti-α1D-AR (1:200), anti-AT1R (1:200), anti-AT2R (1:200), and anti-GAPDH (1:200) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C overnight, followed by incubation with goat anti-rabbit fluorescent (IRDye-conjugated) secondary antibodies (1:10,000; Rockland Immunochemicals, Gilbertsville, PA, USA) for 2 h at room temperature. The images were quantified by the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE, USA). Levels of proteins were normalized to that of GAPDH.
7
Renal Oxidative Stress and Inflammation
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The expression of AT1R, NF-κB p65 and phosphorylation of IKKα/β, IκBα, oxidative stress markers (NOX2, iNOS), and GAPDH in the renal cortex were determined by Western blotting. The renal cortices were homogenized in ice-cold lysis buffer (PBS with 1 % NP40, 1 mmol/L EDTA, 1 mmol/L PMSF, 10 μg /ml leupeptin and 10 μg/ml aprotinin inhibitor). Equal amounts of total extracted proteins (50 μg) were separated on SDS-PAGE and were transferred onto nitrocellulose membranes (Amersham Life Science, Arlington, TX). The blots were subjected to immunoblot analyses with the primary polyclonal antibodies for rabbit anti-AT1R, anti-IKKα/β, phospho-IKKα/β, anti-IκBα, phospho-IκBα, NOX2 and iNOS (1:300; Santa Cruz Biotechnology, Santa Cruz, CA), anti-NF-κB p65 (1:400; BD Transduction Laboratory, Minneapolis, MN, USA), anti-Histone and anti-GAPDH (1:500, Santa Cruz Biotechnology). Immunodetection was accomplished by incubating the blots in horseradish peroxidase-conjugated anti-rabbit secondary antibody (1:10,000 dilution). The bands were visualized using enhanced chemiluminescece kit (Amersham, Arlington, TX), and the band intensities were quantified by densitometry using Quantity-One software (Bio-Rad, Hercules, CA), and normalized with GAPDH expression.
8
Phosphorylation and Expression Analysis
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Total proteins extracted from the hearts or cells were subjected to Western blot analysis for phosphorylation of ERKs, total ERKs, -Actin or GAPDH. The amounts of LOX-1 and AT 1 -R were examined after dividing the membrane fraction and the cytosolic fraction. Following antibodies were used to detect protein expression: anti phosphorylated ERKs (Cell Signaling Technology), anti total ERKs (Santa Cruz Biotechnology), anti LOX-1 (Abcam), anti AT 1 -R (Santa Cruz Biotechnology).
9
Angiotensin II Signaling Pathway Analysis
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Dulbecco modified Eagle medium (DMEM), trypsin and fetal calf serum (FCS) were purchased from Hyclone (South Logan, UT). The anti-Egr-1, anti-AT1R, anti-AT2R and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-phospho-p38, anti-p38, anti-phospho-JNK, anti-JNK, anti-phospho-ERK, anti-ERK and anti-mouse/anti-rabbit conjugated antibodies were from Cell Signaling Technology (Beverly, MA). The 15d-PGJ 2 was purchased from Cayman Chemical (Ann Arbor, MI). The Ang II, Eprosartan (inhibitor of AT1R), PD123319 (inhibitor of AT2 receptor), GW9662 (antagonist of PPAR-γ) and N-acetylcysteine (NAC, inhibitor of ROS production) were obtained from Sigma-Aldrich (St Louis, MO). SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor) and PD98059 (ERK inhibitor) were obtained from Calbiochem (La Jolla, CA). All other chemicals were from Sigma-Aldrich (St Louis, MO), unless specified otherwise.
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