Freshly isolated myocardium, including the LAD, was homogenized in modified RIPA buffer by sonication (2 × 10 pulses, 30% output power) followed by freezing on dry ice for approximately 1 h and then sonication again. Residual cellular debris was removed by centrifugation (18,000 × g for 15 min at 4°C). Protein concentration measured using the Bio-Rad DC kit; 50 μg of total protein was used for immunoprecipitation (IP), and 25 μg was used as an input control. Each IP reaction was adjusted to a total volume of 300 μl in microfuge tubes by the addition of lysis buffer containing protease and phosphatase inhibitors. Finally, 2 μl of mouse monoclonal anti-ET-1 antibody (#ab2786, abcam, 2.5 μg/μl) was added. Tubes were incubated for approximately 15 h at 4°C on a rotor. The next day, 50 μl of a 50:50 slurry of Protein A/G Sepharose beads resuspended in lysis buffer was added to each IP reaction for 1.5 h. The beads were resuspended in LDS buffer that contained 50 mM DTT. The resulting co-IP reaction and input controls were analyzed as described in the ‘Western blot analysis’ section using rabbit-anti-ETA antibody (#9780, Sigma, 1:500) as the primary antibody and donkey-anti-rabbit antibody (NAV931, GE Lifescience, 1:40.000) as the secondary antibody. Blots were developed using ECL Select followed by LAS-4000 image capture.
Ab2786
Manufactured by Abcam
Sourced in United States
Ab2786 is a laboratory reagent used for protein detection and analysis. It is a highly specific antibody that binds to a target protein, allowing for its identification and quantification. The core function of Ab2786 is to facilitate protein-based assays and experiments, but its intended use is not specified.
Automatically generated - may contain errors
Lab products found in correlation
Ab92486, by Abcam (4 mentions)
Ab14184, by Abcam (2 mentions)
Ab8101, by Abcam (2 mentions)
Image pro plus software, by Media Cybernetics (2 mentions)
Ab134047, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ab8245, by Abcam (2 mentions)
Ab1549, by Merck Group (2 mentions)
Dc kit, by Bio-Rad (1 mentions)
Ab2105, by Abcam (1 mentions)
Ab92486, by Santa Cruz Biotechnology (1 mentions)
Ab32572, by Santa Cruz Biotechnology (1 mentions)
Anti mouse, by Jackson ImmunoResearch (1 mentions)
Anti rabbit, by Jackson ImmunoResearch (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Alexa fluor secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab7202, by Abcam (1 mentions)
A2547, by Merck Group (1 mentions)
Vectastain abc kit, by Vector Laboratories (1 mentions)
Ab68418, by Abcam (1 mentions)
20 protocols using ab2786
1
Immunoprecipitation of Endothelin-1 from Myocardium
2
Quantifying RNA, miRNA, and Protein Levels
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RNA was extracted from cultured cells by using TRIzol. For serum samples, RNA was isolated by using TRIzol LS from 200 μl serum. Cel‐miR‐39 was added at 2 nM as a spike‐in control. Relative mRNA or miRNA expression was determined by using SYBR Green (Bio‐Rad) or TaqMan probe‐participated qPCRs, GAPDH and U6 served as internal controls for normalization of mRNA and miRNA expression, respectively. The sequences for qPCR primers are listed in Appendix Table S2 . Proteins were isolated by using RIPA lysis buffer and separated by SDS–PAGE. After being transferred to PVDF membranes, membranes were incubated with various antibodies as indicated. Protein bands were detected by electrochemiluminescence with horseradish peroxidase‐labeled secondary antibodies. The used primary antibodies were as follows: TGF‐β (Abcam, ab92486, 1:1,000); TGFBR2 (Santa Cruz, sc‐17792, 1:500); β‐catenin (Abcam, ab32572, 1:1,000); CTGF (Santa Cruz, sc‐365970, 1:1,000); IL‐1β (Abcam, ab2105, 1:1,000); and ET‐1 (Abcam, ab2786, 1:1,000). Secondary antibodies used were as follows: anti‐mouse (Jackson, 515‐035‐003, 1:5,000) and anti‐rabbit (Jackson, 111‐035‐045, 1:5,000).
3
Protein Expression Analysis in Tissue Sections
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Protein expression was determined on frozen, de-paraffinized sections using antibodies directed against TRPV1, CGRP (GP14100, RA24112; Neuromics, Edina, MN), SP, and ET-1 (ab14184, ab2786; Abcam, Cambridge, MA). Following primary antibody incubation, tissue sections were incubated with the appropriate Alexa-Fluor secondary antibodies (Life Technologies, Grand Island, NY). Sections receiving only secondary antibody were included as negative controls. Sections of spinal cord and dorsal root ganglia (DRGs) were also performed as positive controls for TRPV1, CGRP, and SP staining.
4
Immunohistochemical Analysis of Fibrosis Markers
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Formaldehyde-fixed and paraffin-embedded sections were cut at a thickness of 5 μm. The sections were incubated in 3% H2O2 for 10 min to block the endogenous peroxidase activity and then incubated overnight at 4 °C with primary antibodies against MCP-1 (ab7202, 1:100 dilution; Abcam, Cambridge, UK), α-SMA (A2547, 1:200 dilution; Sigma, St. Louis, MO, USA), and ET-1 (ab2786, 1:100 dilution; Abcam, Cambridge, UK). The negative control was incubated with normal serum instead of primary antibodies. All the sections were immunostained with the VECTASTAIN ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA) in accordance with the manufacturer’s specifications.
5
Quantification of RNA and Protein Levels
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Total RNA and miRNA were extracted from serum samples, CD31+ microparticles, HUVECs, and HASMCs by the use of TRIzol reagents (Invitrogen). The relative level of mRNA or miRNA was determined by using SYBR Green or TaqMan probe (Takara). For serum samples, cel-miR-39 (2 nM) was added as a spike-in control. GAPDH and U6 were used as internal controls for normalizing miRNA and mRNA expression. A comparative Ct method, X = 2−ΔΔCt, was used to determine the relative gene expression [25 (link)]. The sequences for real-time PCR primers are presented in Supplemental Table 4 . For immunoblotting, proteins were isolated from HUVECs or HASMCs using the RIPA lysis buffer, separated by 4-10% SDS-PAGE, and then transferred to PVDF membranes. After blocking with 5% milk in TBST, the membranes were incubated with the primary antibodies for ACE1 (ab28311, Abcam), TGF-β (ab92486, Abcam), CTGF (sc-365970, Santa Cruz), ET-1 (ab2786, Abcam), β-actin (sc-47778, Santa Cruz), CD81 (ab79559, Abcam), and CD63 (ab68418, Abcam). The β-actin blot was the loading control. The protein bands were observed by enhanced chemiluminescence (Millipore).
6
Evaluating ET-1 and MCP-1 Responses in H2O2-Treated HUVECs
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HUVECs were treated with H2O2 (400 μM) in the presence of or no GTs (500 μg/ml, 1 h pretreatment) for 5 h. Treated cells were fixed and permeabilized with 0.1% Tween 20 in PBS washing buffer for 30 min at room temperature. Cells were then stained with anti-ET-1 (Abcam ab2786, 1 : 200) or anti-MCP-1 (Abcam ab8101, 1 : 200) antibodies.
7
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.
8
Quantifying Eosinophils and ET-1 in Lung Tissue
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Lung tissue was fixed in formaldehyde solution and embedded in paraffin. The tissue was sectioned at a thickness of 4–5 μm, and stained with H&E solution for analyzing the difference between eosinophils [29 (link)]. For immunohistochemistry, the paraffin sections were rehydrated and transferred to 100% methanol with 3% hydrogen peroxide to eliminate endogenous peroxidase activity. Sections were then rinsed with PBS, and blocked with blocking serum before incubation with a primary antibody against ET-1 (ab2786, abcam, 1:250 in antibody diluent) overnight. Control sections were incubated with PBS instead of the primary antibody. A systematic sampling method was used to evaluate random, non-overlapping calibrated fields for each variable, and the terminal bronchioles were used as the independent landmark for selecting a small pulmonary arteriole [29 (link),30 (link)]. We used Image Pro Plus software (Media Cybernetics) to make the measurements. Tissue sections were analyzed by one without knowledge of the group from which the tissue was taken. At least eight slides from each lung were taken and analyzed.
9
Vascular Remodeling and Inflammation in Carotid Artery Injury
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Mice were euthanized 3, 14, or 17 days after surgery. LCAs and right common carotid arteries (RCAs) were harvested and cryopreserved in OCT compound. Eight micrometer cryosections of the artery tissue from the segment between 1.5 and 2 mm from the ligature were stained with hematoxylin and eosin (H&E) or TUNEL staining (Millipore). For immunofluorescence staining, sections of arterial tissue were incubated with the antibodies as indicated, including anti-CD31 (Abcam ab28364, 1 : 50), anti-F4/80 (Abcam ab6640, 1 : 100), anti-endothelin-1 (ET-1) (Abcam ab2786, 1 : 250), anti-von Willebrand factor (vWF) (Abcam ab68545, 1 : 50), and anti-monocyte chemoattractant protein-1 (MCP-1) (Abcam ab8101, 1 : 20). The number of cells displaying specific staining was scored in a blinded manner.
10
Protein Expression Analysis in Cells
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RIPA lysis buffer (Beyotime, Shanghai, China) was applied to extract protein and the protein concentration was estimated using bicinchoninic acid method. Protein extracts were separated by SDS-PAGE and then transferred to PVDF membranes (Beyotime, Shanghai, China). Membranes were incubated with primary antibodies against PTPRO (Abcam, ab231560, 1:1000), MMP2 (Abcam, ab181286, 1:1000), MMP9 (Abcam, ab228402, 1:1000), Ki67 (Abcam, ab16667, 1:1000), VEGF (Abcam, ab214424, 1:1000), ET-1 (Abcam, ab2786, 1:1000), sFIt-1 (Abcam, ab32152, 1:1000), ERp44 (Abcam, ab137611, 1:3000) and GAPDH (Abcam, ab181602, 1:10,000) overnight at 4°C. On the second day, membranes were exposed to a horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 h. Protein signals were developed with an enhanced chemiluminescence (ECL) detection kit (Beyotime, Shanghai, China). Protein expression was analyzed using Image J software with GAPDH as a loading control.
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