Equivalent amounts of cell lysates (20 μg protein/lane) were loaded onto 10% SDS-PAGE, proteins separated, and proteins transferred to nitrocellulose membranes. The membranes were blocked with 5% bovine serum albumin followed by overnight incubation at 4°C with respective ET receptor antibodies (anti-ETA receptor; ab85163, Abcam or anti-ETB receptor; AER002, Alomone Labs) or senescence marker proteins p16INK4a (PA1–30670, Fisher scientific), p21 (AHZ0422, Invitrogen), cyclin D1 (92G2, Cell Signaling), lamin A/C (2032, Cell Signaling) or membrane integrity marker proteins occludin1 (OC-3F10, Invitrogen), claudin5 (35–2500, Invitrogen), p53 (DO-7, Invitrogen) VE-cadherin (2158, Cell Signaling) at 1:1000 dilution or anti-β-actin at 1:20000 dilutions. After washing, membranes were incubated for 1 hour at 20°C with appropriate secondary antibodies (horseradish peroxidase [HRP]-conjugated; dilution 1:5000). Pre-stained molecular weight markers were run in parallel to identify the molecular weight of proteins of interest. For chemiluminescent detection, the membranes were treated with enhanced chemiluminescent reagent and the signals were monitored on Amersham imager 680 (GE Healthcare Bio-Sciences Corp., Marlborough, MA). Relative band intensity was determined by densitometry on Image-J and normalized with β-actin protein.
Ab85163
Manufactured by Abcam
Sourced in United Kingdom
Ab85163 is a laboratory reagent that can be used in various experimental procedures. The product details and intended use are not available for this item.
Automatically generated - may contain errors
Lab products found in correlation
Aer002, by Alomone (3 mentions)
Ab117529, by Abcam (3 mentions)
Enhanced chemiluminescence reagent, by Beckman Coulter (3 mentions)
Polyvinylidene uoride membrane, by Merck Group (3 mentions)
Ab8245, by Abcam (3 mentions)
Amersham imager 680, by Cytiva (2 mentions)
Oc 3f10, by Thermo Fisher Scientific (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
Ve cadherin, by Cell Signaling Technology (1 mentions)
Ab2786, by Abcam (1 mentions)
Chemiluminescent reagent, by Merck Group (1 mentions)
Ab84593, by Abcam (1 mentions)
Ab133303, by Abcam (1 mentions)
Bq123, by Merck Group (1 mentions)
Bq788, by Merck Group (1 mentions)
Cay10650, by MedChemExpress (1 mentions)
Af1015, by Affinity Biosciences (1 mentions)
Af0155, by Affinity Biosciences (1 mentions)
13 protocols using ab85163
1
Western Blot Analysis of Senescence and Membrane Markers
2
Placental Protein Expression Analysis
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On GD21, placental tissue proteins were first extracted, followed by measuring the concentration of the proteins using the BCA kit (Pierce™ BCA, Thermo Fisher Scientific). Proteins were loaded onto a 10% polyvinylidene fluoride membrane (Merck Millipore, Burlington, MA, USA). Then, the samples were blocked using 5% milk for 1 h, followed by incubated using primary antibodies for ET-1 (1:1000, ab2786, Abcam, Shanghai, China) and ETAR (1:500, ab85163, Abcam, Shanghai, China) overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies were used to incubate temperature for 1 h at room temperature. Finally, chemiluminescent reagents (Merck Millipore, Burlington, MA, USA) were used to detect the signals. Image J was used for quantifying the signals.
3
Signaling Pathways Regulating Endothelin
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Endothelin receptor type A (ETRA) antagonist BQ123 and endothelin receptor type B (ETRB) antagonist BQ788 were purchased from Sigma (St. Louis, MO, USA). ET-1 (human, porcine) was purchased from Tocris Bioscience (Minneapolis, MN, USA). The NOX4 inhibitor GLX351322, secreted phospholipase (sPLA2) inhibitor varespladib, cytosolic PLA2α (cPLA2α) inhibitor CAY10650, ROS inhibitor N-Acetyl-D-cysteine (NAC), Src antagonist Src inhibitor 1, extracellular signal-regulated kinase (ERK) antagonist PD98059, and protein kinase B (Akt) antagonist LY294002 were purchased from Med Chem Express (Monmouth Junction, NJ, USA). Anti-NOX4 and anti-GATA4 antibodies were obtained from Abcam (ab133303, ab85163, ab117529, and ab84593, respectively; Cambridge, UK). The anti-Src antibody was from Millipore (04-889; Burlington, MA, USA). Anti-NOX2 antibody was obtained from Bioss (bs-3889R; Beijing, China). Anti-Akt, phospho-Akt (S473), and β-actin antibodies were acquired from Bioworld Technology (BS1008, BS4006, and AP0060, respectively; Nan Jing, China). Anti-ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), cPLA2, and phosho-cPLA2 (Ser505) antibodies were obtained from Affinity (AF0155, AF1015, AF6329, and AF3329, respectively; Chang Zhou, China).
4
Quantitative Analysis of Endothelin Pathway
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Lung tissues were homogenized and centrifuged at 12000 rpm for 10 min. Supernatants were extracted and the protein concentration was determined with a BCA kit (G2026; Servicebio). Protein solution was added into the loading buffer (G2013; Servicebio) in a ratio of 4:1 and denatured in boiling water for 15 min. Samples were loaded on 5% SDS-PAGE gels (Servicebio), then separated by electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, United States). After being blocked in 5% fat-free milk for 1 h, membranes were incubated overnight with the following primary antibodies: ET-1 (ab117757; Abcam, Cambridge, United Kingdom), ET-A (ab85163; Abcam), ET-B (ab262694; Abcam), ECE-1 (sc-376017; Santa Cruz Biotechnology, Dallas, TX, United States), and GAPDH (ab9485; Abcam). The next day, the membranes were incubated in horseradish peroxidase-conjugated secondary antibodies, which were diluted 3000 times with Tris-buffered saline Tween-20. Bands were visualized with ECL solution (Servicebio) and the band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MA, United States).
5
Western Blot Analysis of Aortic Proteins
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Proteins were first extracted from each mid-aorta lysate (20 μg), separated on 10% SDS-PAGE gels, and then transferred to polyvinylidene fluoride (PVDF) microporous membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk, and immunoblotted with the following primary antibodies: RhoA (1 : 1000, 10749–1-AP, Protein-tech, Wuhan, China), ROCK I (1 : 1000, ab134181, Abcam, Cambridge, UK), ROCK II (1 : 20000, ab125025, Abcam), MLCP (1 : 1000, ab32519, Abcam), endothelin A receptor (ETA, 1 : 900, ab85163, Abcam), and collagen I (Col I, 1 : 500, ab6308, Abcam). The membranes were incubated with horseradish peroxidase (HRP)-conjugated antirabbit or antimouse secondary antibodies (1 : 15000, Protein-tech), and then subjected to chemiluminescence detection using the ECL kit (NcmECL Ultra, NCM Biotech, Suzhou, China). Intensity of immunoreactive bands was normalized using GAPDH expression (1 : 7000, 10494-1-AP, Proteintech), and quantified using densitometry analysis performed with the Image J software (Version 1.53e, NIH, Bethesda, Maryland, USA).
6
Immunoblotting Analysis of ETA and ETB Receptors
7
ETAR Protein Level Detection in Bladder Tissue
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Western blot analysis was carried out for the detection ETAR protein levels. Bladder tissues were thoroughly grinded under liquid nitrogen and protein samples were extracted using RIPA buffer with 1 mM PMSF (Beyotime Biotech, Shanghai, China). BCA Kit (Beyotime Biotech, China) was used to measure protein concentration. Sodium dodecyl sulphate-polyacrylamide gel was applied to analyze 30 μg of protein from each group and then transferred to a polyvinylidene fluoride membrane. After the primary antibodies ETAR (ab85163, Abcam, MA, USA) and GAPDH (60004-1-Ig, Proteintech, Wuhan, China) and appropriate Secondary antibody reactions, the blot membranes were processed by using an enhanced chemiluminescence (ECL) kit (Immobilon, Sigma-Aldrich), followed by film exposure and densitometry analysis.
8
Western Blot Analysis of ETA and ETB Receptors
9
Protein Quantification in Tissues and Cells
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Total protein in tissues, cells and exos was obtained by radio-immunoprecipitation assay lysate containing protease inhibitors, separated by 10% sodium lauryl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene uoride membrane (Millipore, MA, USA) and sealed with 5% skim milk. The corresponding primary antibodies EDNRA (1:1000, ab85163) and GAPDH (1:1000, ab8245, Abcam), as well as the corresponding secondary antibody were incubated with the membrane.
The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
10
Quantitative Protein Analysis via RIPA
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Total protein in tissues, cells and exos was obtained by radio-immunoprecipitation assay lysate containing protease inhibitors, separated by 10% sodium lauryl sulfate polyacrylamide gel electrophoresis, transferred to polyvinylidene uoride membrane (Millipore, MA, USA) and sealed with 5% skim milk. The corresponding primary antibodies EDNRA (1:1000, ab85163) and GAPDH (1:1000, ab8245, Abcam), as well as the corresponding secondary antibody were incubated with the membrane. The enhanced chemiluminescence reagent (Beckman Coulter) was used to quantify the protein [26] .
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