Three sections from each graft were stained with HE and the thickness was measured. Immunohistochemistry and immunofluorescence was performed as described previously [16] . The sections were incubated overnight with anti-ET A R (Abcam, ab85163) and anti-ET B R (Abcam, ab117529) for immunohistochemistry. To evaluate the expression of ET B R in endothelial and smooth muscle cells, the sections were double stained by anti-CD 31(Abcam, ab119339) and anti-α-SMA (Abcam, ab28052) with anti-ET B R (Abcam, ab117529) by immunofluorescence. Negative controls were included using isotypematched control antibodies (Abcam). In the control experiments, either the primary antibody or secondary antibody was omitted. The stained arterial segments were observed under a confocal microscope (Nikon, C1plus, Nikon Instruments, Inc, NY, USA) and analyzed by IMAGE-PRO PLUS. For quantification, the mean optical densities of the ET A R and ET B R staining in the immunohistochemistry images were determined using IMAGE-PRO PLUS. In each section, the optical density was measured at 4 preset areas, and the mean optical density was obtained from 6 vessels.
Ab117529
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab117529 is a primary antibody used for research purposes. It is a rabbit monoclonal antibody directed against a specific target. The core function of this product is to detect and bind to the target antigen, enabling its identification and analysis in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Ab117521, by Abcam (3 mentions)
Ab85163, by Abcam (3 mentions)
Protease inhibitor, by Boster Bio (1 mentions)
Sds page, by Beyotime (1 mentions)
Enhanced chemi luminescence (ecl), by Abbkine (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Goat anti mouse igg hrp, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Control sirna sc 37007, by Santa Cruz Biotechnology (1 mentions)
Endothelin 3, by Merck Group (1 mentions)
Pcmv6 entry vector, by OriGene (1 mentions)
Ab9485, by Abcam (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)
9 protocols using ab117529
1
Quantifying Endothelin Receptor Expression
2
Western Blot Analysis of ETAR and ETBR
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Total cell protein was extracted on ice with RIPA lysis buffer (Beyotime biotechnology, Shanghai, China) in the presence of freshly added protease inhibitors (Boster Biological Technology, Wuhan, China), and quantified by the BCA assay (Pierce, Cramlington, UK). A total of 30 μg protein from each sample was subjected to 10% SDS–PAGE (Beyotime biotechnology, Shanghai, China) by electrophoresis under reducing conditions and transferred to PVDF membrane (Millipore Corporation, Billerica, MA, USA). The blotted membrane was then blocked with 5% skim milk for 1 h at room temperature and incubated respectively with anti-ETAR (1:5000, ab117521, Abcam, Cambridge, MA), ETBR (1:5000, ab117529, Abcam, Cambridge, MA) and anti-GAPDH (1:2000, 10494-1-AP, ProteinTech, Chicago, USA) antibody overnight at 4°C. The membranes were further incubated with HRP-conjugated anti-rabbit secondary antibodies and detected using the enhanced chemiluminescence (ECL; Abbkine, Redlands, CA, USA) method. The densitometry was performed using Image J software.
3
Endothelin Receptor B Signaling Pathway
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Endothelin 3 was purchased from Sigma Aldrich and reconstituted to 1 mg/ml in water. siRNA specific for EDNRB (sc-39962), along with control siRNA (sc-37007) were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). EDNRB antibodies for Western blotting (ab117529) and flow cytometry (ab129102) and GAPDH antibody for Western blotting (ab9485) 31 (link) were purchased from Abcam (Cambridge, United Kingdom). pERK (#4370) and ERK (#4695) 32 (link) antibodies, along with pan AKT (4691), phospho AKT T308 (13038) and phospho-AKT1 S473 and phospho-AKT2 S474 (8599) were all purchased from Cell Signaling (Cell Signaling Technology, Beverly, MA). Secondary antibodies, including goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, goat anti-rabbit IgG-FITC were all purchased from Cell Signaling. Plasmid expression vectors encoding EDNRB-442 (RG 232943), EDNRB-536 (RC 216462), and EDNRB-436 (RC 225723) and accompanying empty vector controls (pCMV6-Entry vector) were purchased from Origene (Rockville, MD).
4
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).
5
Immunofluorescence Analysis of ETB Receptor
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Anti-SM22 (1:100, ab10135, Abcam, UK), Goat isotope control IgG (5 μg/mL, ab18433 Abcam), Rabbit anti-ETB receptor (1:100, ab117529, Abcam), Rabbit isotope control IgG (10 μg/mL, ab18433, Abcam), Allophycocyanin (APC)-conjugated donkey anti-rabbit IgG (1:100, Jackson ImmunoResearch), Alexa 488-conjugated donkey anti-goat IgG (1:100, Jackson ImmunoResearch), Fixable Viability Dye eFluor 780 (eBioscience), Liberase TM Research Grade (Roche), German coverslip with Poly-L-lysine coating on both sides cat no H-12-pII Neuvitro, USA, Vectashield Antifade Mounting Medium (Vector Laboratories, USA), BSA (Bovine Serum Albumin, Sigma Aldrich), FBS (Fetal Bovine Serum, Sigma Aldrich), Triton X-100 (Sigma Aldrich).
6
Western Blot Analysis of Endothelin Receptors
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For western blot analysis, total proteins were extracted from radioimmunoprecipitation assay (RIPA) lysis buffer (Solarbio, R0020). Protein was quantified by a bicinchoninic acid assay (BCA) protein assay kit (Beyotime, P0012S). Total protein was denatured by boiling for 10 min. Protein (40 µg) was separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, CA, USA) and transferred to a nitrocellulose filter membrane (Millipore, Massachusetts, USA). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with the following primary antibodies: rabbit polyclonal anti-endothelin A Receptor antibodies (Abcam, ab178454, 1:1000 in 5% skim milk), rabbit polyclonal anti-Endothelin B Receptor antibodies (Abcam, ab117529, 1:1000 in 5% skim milk), and rabbit polyclonal anti-GAPDH (Abcam, ab37168, 1:1000 in 5% skim milk) for 12 h to 16 h. Then, peroxidase-conjugated AffiniPure goat anti-rabbitIgG(H + L)(ZSGE-BIO, ZB-2301, 1:10,000 in 5% skim milk) was added for 2 h at room temperature and subsequently developed using an enhanced chemiluminescence reagent (Beyotime, P0018A). The signals emitted for chemiluminescence were detected using the Image Quant LAS 4000 (Bio-Rad, CA, USA, 00746947) and analysed with ImageJ software (NIH). The results were normalized to the corresponding densitometry signal of GAPDH.
7
Renal Artery Endothelin Receptor Localization
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Renal arteries were fixed in 4% paraformaldehyde, washed, dehydrated by soaking in a graded ethanol series (75, 85, 90, 95 and 100%) and cleared in xylene. Vessels were paraffin-embedded and sliced into 5-μm sections at a 200-μm interval from the distal (kidney) to proximal (abdominal aorta) region for hematoxylin and eosin (HE) staining. For immunohistochemistry, sections were blocked in 3% bovine serum albumin (BSA) for 30 min and then incubated overnight at 4 °C with the primary antibody anti-ETAR (1:500; ab117521, Abcam, Cambridge, UK) or anti-ETBR (1:500; ab117529, Abcam, Cambridge, UK). Subsequently, the sections were washed, incubated with HRP-labeled goat anti-rabbit secondary antibody for 50 min at room temperature. The immunoreactions were developed with a diaminobenzidine (DAB) chromogenic kit. The nuclei were counterstained with HE.
8
Endothelin-1 and TGF-β1 Signaling Pathways
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Antibodies used were α-smooth muscle actin (α-SMA) (ab7817; Abcam, Cambridge, UK), pSmad3 (ab51451; Abcam), Smad3 (ab40854; Abcam), VEGF (ab1316; Abcam), cleaved caspase-3 (#9661; Cell Signaling Technology, Danvers, MA, USA), ET-1 (ab117757; Abcam), CD31 (sc-1506; Santa Cruz Biotechnology, Santa Cruz, CA, USA), ETRA (ab117521; Abcam), ETRB (ab117529; Abcam), GAPDH (#5174; Cell Signaling Technology), anti-rabbit HRP-linked IgG (#7074; Cell Signalling Technology) and anti-mouse IgG HRP-linked antibody (#7076; Cell Signaling Technology). For fluorescence microscopy, we used goat or donkey secondary antibody conjugated with Alexa Fluor-488 and Alexa Fluor-555 (Abcam). Human rET-1 (100-21; PerproTech, London, UK) and human rTGF-β1 (240-B; R&D Systems, Minneapolis, MN, USA) were used in vitro to treat cells.
9
Quantifying Aortic Protein Expression
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The aortas were frozen in liquid nitrogen and homogenized in a cell extract denaturing buffer that contained a phosphatase inhibitor cocktail and protease inhibitor cocktail. The cultured cells were resolved using RIPA Lysis Buffer. The antibodies for ET A R (Abcam, ab85163), ET B R (Abcam, ab117529), phospho-NF-κB p65 (Abcam, ab86299), and NF-κB p65 (Abcam, ab16502) were used as primary antibodies. Secondary antibodies were HRP-labeled goat anti-rabbit IgG or rabbit anti-goat IgG (Biosharp). All antibodies were used at a dilution of 1:1000, and the experiments were repeated 3 times. An anti-GAPDH antibody (Bioworld, AP0063) was used for normalization. The western blot band intensity was determined using IMAGE-PRO PLUS.
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