Antibodies against various proteins were obtained from the following sources: ACTG2 (ab189385, Abcam), ACTA2 (ab5694, Abcam), MYH2 (ab124937, Abcam), MYH7B (ab172967, Abcam), HISTH2B (ab52599, Abcam), HIST1H2BM (SAB1301739, Sigma), and β-actin (A1978, Sigma). Commercially available horseradish peroxidase (HRP)-conjugated secondary antibodies (7074, 7076) were obtained from Cell Signaling Technology. All other chemical reagents were procured from Sigma Chemical Corp.
Ab172967
Manufactured by Abcam
Sourced in United States
Ab172967 is a lab equipment product. It is a device designed for use in laboratory settings. The core function of this product is to perform specific tasks or measurements as part of scientific research or analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab19645, by Abcam (4 mentions)
Ab180649, by Abcam (2 mentions)
Ab225844, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab124937, by Abcam (1 mentions)
Ab52599, by Abcam (1 mentions)
Ab189385, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Ab5694, by Abcam (1 mentions)
Ab5589, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Ab47285, by Abcam (1 mentions)
Ab110413, by Abcam (1 mentions)
Ab463, by Abcam (1 mentions)
Ab40742, by Abcam (1 mentions)
Murf1, by Santa Cruz Biotechnology (1 mentions)
Sc 33782, by Santa Cruz Biotechnology (1 mentions)
Ab46154, by Abcam (1 mentions)
Alpha innotech fluorchem hd2, by Thermo Fisher Scientific (1 mentions)
11 protocols using ab172967
1
Immunoblotting Antibody Validation
2
Western Blot Analysis of Cardiac Proteins
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Cardiac muscle (25–50 mg) was homogenized as previously reported [22 (link)] and 5μg of homogenate loaded for SDS-PAGE. Proteins were separated on a 6%, 7.5%, 10% or 12% resolving gel as required to optimize for MW separation, and transferred to polyvinylidene difluoride membrane (Roche, Laval, QC, CA). The following commercially available antibodies were used: total OXPHOS antibody cocktail (Abcam, Cambridge, MA, USA, ab110413, 1:500,), eNOS (Abcam, ab5589, 1:1000), VEGF (Abcam, ab46154, 1:1000), HIF1α (Abcam, ab463, 1:1000), alpha tubulin (Abcam, ab40742, 1:5000), muscle RING finger protein-1 (MuRF1; Santa Cruz Biotechnology, Dallas, TX, USA, sc-32920, 1:500), Muscle atrophy F-box (MAFbx; Santa Cruz, sc33782, 1:500), forkhead transcription factor-3a, Serine residue 253 (FOXO3a Ser253; Abcam, ab47285, 1:500), atrial natriuretic peptide (ANP; Abcam, ab180649, 1:500), BNP (Abcam, ab19645, 1:500) and beta-myosin heavy chain (β-MHC; Abcam, ab172967, 1:2000). All samples were detected from the same Western blot by cutting gels and transferring onto a single membrane to limit variability. Equal loading of protein was verified using Ponceau staining as well as constant alpha tubulin. All blots were quantified using enhanced chemiluminescence (Perkin Elmer, Woodbridge, ON, CA) and quantified by densitometry (Alpha Innotech Fluorchem HD2, Fisher Scientific, Ottawa, ON, CA).
3
Protein Extraction and Western Blot Analysis
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Protein was extracted from frozen portions of the LV by using RIPA buffer (BP-115D, Boston BioProducts, Ashland, MA) and a 2 ml glass homogenizer. Protein lysates were quantified using a BCA protein assay kit (23227, Thermo Fisher). About 30 μg of proteins loaded onto 10 or 12% SDS PAGE gels.
All membranes were blocked using 5% nonfat dry milk in Tris-buffered saline (TBS) at room temperature for 30 min. The primary antibodies used were: β-MHC (1:1,000, ab172967, Abcam), CSE (1:1500, H00001491-M02, Abnova), c-Fos (1:1,000, 2250 s, Cell Signaling), total c-Jun (1:1,000, 9165 s, Cell Signaling), MMP9 (1:1,000, ab119906, Abcam), and TGF-β1 (1:100, sc146, Santa Cruz) for 1 h. Secondary antibody staining was performed using: anti-mouse IgG-HRP, and anti-rabbit IgG-HRP (used at half the concentration of the respective primary antibody, Cell Signaling). Restore Stripping Buffer (46430, Thermo Fisher) was used as necessary. Protein expression was normalized against total protein measured by Ponceau staining. Bands were visualized with a ChemiDoc Imaging System (Bio-Rad), and band intensity was analyzed by ImageLab software (Bio-Rad).
All membranes were blocked using 5% nonfat dry milk in Tris-buffered saline (TBS) at room temperature for 30 min. The primary antibodies used were: β-MHC (1:1,000, ab172967, Abcam), CSE (1:1500, H00001491-M02, Abnova), c-Fos (1:1,000, 2250 s, Cell Signaling), total c-Jun (1:1,000, 9165 s, Cell Signaling), MMP9 (1:1,000, ab119906, Abcam), and TGF-β1 (1:100, sc146, Santa Cruz) for 1 h. Secondary antibody staining was performed using: anti-mouse IgG-HRP, and anti-rabbit IgG-HRP (used at half the concentration of the respective primary antibody, Cell Signaling). Restore Stripping Buffer (46430, Thermo Fisher) was used as necessary. Protein expression was normalized against total protein measured by Ponceau staining. Bands were visualized with a ChemiDoc Imaging System (Bio-Rad), and band intensity was analyzed by ImageLab software (Bio-Rad).
4
Western Blot Analysis of Cardiac Markers
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Cells were lysed using a lysis buffer (Beyotime, China) and the lysates were separated on 10% SDS-PAGE. The proteins on the gel were then transferred onto a PVDF membrane. After blocking with 5% nonfat dry milk, the membranes were incubated with primary antibodies (1 : 1000, anti-cTnT, ab8295; anti-NKX2.5, ab106923; anti-GATA4, ab86371; anti-α-MHC, ab50967; anti-β-MHC; ab172967, Abcam; anti-SRF, MAB4369; anti-GAPDH, ABS16, Merck Millipore) overnight at 4°C. Membranes were washed and incubated with corresponding HRP-labeled secondary antibodies (anti-mouse IgG, 1: 10000, 71045; anti-rabbit IgG, 1 : 10000, AP188P, Merck Millipore). The band signals were visualized using the ECL Western Blotting substrate (Pierce) and the signal intensity was quantified using ImageQuant 5.2 (GE Healthcare, Piscataway, NJ). To clearly demonstrate the difference, the relative gray-scale value of target protein and internal control of control group was set as 1.
5
Immunofluorescence Characterization of Cardiac Cells
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Immunofluorescence was performed on 10-µm-thick sections. Following permeabilization with PBS containing 0.5% Triton X-100 for 15 min and blocking with 1% BSA in PBS for 60 min at room temperature, the sections were incubated with primary antibodies diluted in PBS containing 1% BSA and 1% Tween 20 at 4 °C overnight. Primary antibodies used in this study include 1:100 CD90 (Abcam #Ab225), 1:100 MYH6/7 (Abcam #Ab50967), 1:100 TNNT2 (Thermo Fisher #MA5–12960), 1:200 ACTN4 (Sigma-Aldrich #A7811), 1:100 GJA1 (Abcam #Ab11370), 1:100 human MYH7 (Abcam #Ab172967), and 1:100 human mitochondria (Abcam #Ab92824). After washing three times with 1% Tween 20 in PBS, the samples were incubated with secondary antibodies for 60 min at room temperature in the dark. Secondary antibodies used in this study include either 1:400 anti-mouse IgG Alexa Fluor 488 (Invitrogen #A10680) or 1:400 anti-rabbit IgG Alexa Fluor 647 (Invitrogen #A21245). After washing again with 1% Tween 20 in PBS, the sections were stained with DAPI solution (VectaShield) for nuclear staining and then mounted on slides. Imaging of heart sections was performed with a Laser Scanning Microscope LSM 880 NLO with Airyscan processing (Zeiss).
6
Antibody-based Cardiac Protein Analysis
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The antibodies were used as follows: Anti-ANP antibody (ab225844, abcam, WB:1:1000), Anti-BNP antibody (ab19645, abcam, WB:1:500), Anti-β-MHC antibody (ab172967, abcam, WB:1:1000), Anti-Ythdf2 antibody (24744–1-AP, proteintech, IF:1:200; WB:1:1000), Anti-CPT-1a (66039-1-Ig, proteintech, IF:1:200, WB:1:1000), Anti-PPARα (AF5301, Affinity, WB:1:1000) GAPDH antibody (abs132004, absin, WB:1:5000), anti-β-tubulin antibody (10094-1-AP, Proteintech, WB:1:1000), goat anti-rabbit IgG-HRP (abs20002, absin, WB:1:10000), goat anti-mouse IgG-HRP (abs20001, absin, WB:1:10000). AngII (HY-13948) was purchased from MCE (China).
7
Immunostaining and Morphometric Analysis of C2C12 Myofibres
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Myofibre immunostaining of C2C12 cells was performed using primary antibodies against Myh (catalogue no.: ab172967; Abcam). Staining for muscle fibre-type was performed using the Muscle Fiber Typing Staining Kit (Servicebio), as per manufacturer’s instructions. Images were captured and processed with a Leica DMi8 automated microscope (Leica), and the myofibre area was measured using ImageJ software (RRID: SCR_003070; version 1.51; NIH) (see ESM Methods ).
8
Western Blotting of Cardiac Proteins
9
Histological Analysis of Tissue Samples
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The paraffin-embedded tissues were sliced and performed with hematoxylin-eosin (HE), Sirius red, or Masson trichrome staining. The interstitial fields and the perivascular fields containing vasculature were imaged. Immunohistochemistry was performed with antibodies of IL-6 (D220828, Sangon, Shanghai, China), TNF-α (bs-2081R, Bioss, Beijing, China), and ANP and MYH7B (ab225844 and ab172967, Abcam, U.S.). The EVOS FL Auto Imaging System (Life Technologies, ThermoFisher Scientific, Waltham, MA, U.S.) was operated to obtain 3-5 random fields of each image.
10
Protein Expression Analysis in NRCMs
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NRCMs or tissue samples were lysed by radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, China). The total protein concentrations were determined by BCA protein assay kit (Beyotime Biotechnology, China). A total of 30 ug proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were blocked with 5% non-fat milk and incubated overnight with the primary antibodies against BNP (1:500, rabbit #ab19645 abcam), β-MHC (1:1,000, rabbit #ab172967 abcam), KLF4 (1:1,000, rabbit #bs-1064R BIOSS), PI3K (1:1,000, rabbit #ab182651 abcam), p-PI3K (1:1,000, rabbit #205841-1-AP Proteintech), AKT1 (1:1,000, rabbit # bs-0115M BIOSS), p-AKT1 (1:1,000, rabbit # bs-0876R BIOSS), β-actin (1:1,000, rabbit #abmart P30002) and GAPDH (1:2,000, rabbit #2118S CST). Then, the membranes were washed and treated with horseradish peroxidase-conjugated secondary antibodies (CST 7074) at room temperature for 2 h. ECL System was employed to examine the protein bands and the intensity of protein bands measured by ImageJ software.
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