Cells were lysed in ice-cold buffer containing 50 mmol/L Tris-HCl, pH 7.4, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1% NP-40, 0.1% sodium deoxycholate, 0.1% SDS, 100 mmol/L NaCl, 10 mmol/L NaF, 1 mmol/L sodium pyrophosphate, 1 mmol/L sodium orthovanadate, 1 mmol/L Pefabloc SC, and 2 mg/mL protease inhibitor cocktail (Roche Diagnostics Corp). Protein concentrations were determined using the DC Protein Assay Kit (Bio-Rad Laboratories). Cell lysates containing 12.5 to 25 μg of protein were analyzed by SDS-PAGE and immunoblotting. Primary antibodies used include the following: Cav-1 (No. 610058; BD Biosciences), LC3B (No. 2775; Cell Signaling), SQSTM1/p62 (No. ab56416; Abcam), and β-actin (No. sc-69879; Santa Cruz Biotechnology), PKB/AKT (protein kinase B, also known as Akt; No. 9272; Cell Signaling), p-AKT (No. 9277; Cell Signaling), pS6K (No. 9205; Cell Signaling), S6K (No. 9202; Cell Signaling), ATG5 (No. 2630; Cell Signaling), connexin-43 (Cx-43; No. ab11370; Abcam), vinculin (No. v9131; Sigma), and HSP90 (No. 610419; BD Biosciences). Secondary antibodies were fluorescence-labeled antibodies (LI-COR Biotechnology). Bands were visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnology). Densitometry analysis was performed with ImageJ software (National Institutes of Health) or Image Studio Lite (LI-COR Biosciences).
Connexin43 cx43
Manufactured by Abcam
Sourced in United Kingdom, United States
Connexin43 (Cx43) is a key structural protein found in gap junctions, which facilitate intercellular communication. It plays a crucial role in the regulation of cell-to-cell signaling and the maintenance of tissue homeostasis.
Automatically generated - may contain errors
Lab products found in correlation
α actinin, by Abcam (3 mentions)
Live dead cell staining kit, by Thermo Fisher Scientific (2 mentions)
Gelatin, by Merck Group (2 mentions)
Alpha smooth muscle actin α sma, by Boster Bio (2 mentions)
F4 80 antibody, by Thermo Fisher Scientific (2 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by Abcam (1 mentions)
Vinculin, by Merck Group (1 mentions)
Image studio lite, by LI COR (1 mentions)
Hsp90, by BD (1 mentions)
Pefabloc, by Roche (1 mentions)
Cav 1, by BD (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
P s6k, by Cell Signaling Technology (1 mentions)
Pkb akt, by Cell Signaling Technology (1 mentions)
Fluorescence labeled antibodies, by LI COR (1 mentions)
6 protocols using connexin43 cx43
1
Cell Lysis and Protein Analysis
2
Immunofluorescence Analysis of Vascular Cell Markers
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The PET membranes were removed from the cell culture inserts on day 8 after the cells had been cultured. Following this, the cells were rinsed with PBS, fixed in paraformaldehyde at a concentration of 4% for 20 minutes at room temperature, and then washed once more with PBS. Nonspecific binding was blocked using 3% bovine serum albumin in PBS for 30 min. Then, membranes were incubated overnight at 4°C with a monoclonal mouse anti-human primary antibody to alpha smooth muscle actin (α-SMA) (1 : 100 dilution) (Santa Cruz Biotechnology, Dallas, TX, USA), calponin (1 : 200 dilution) (Abcam, Cambridge, UK), and connexin43 (Cx43) (1 : 200 dilution) (Abcam) in PBS. After washing with PBS, the membranes were incubated goat anti-mouse FITC-conjugated secondary antibody (1 : 100 dilution) (Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at 37°C, and the nuclei were stained with DAPI (Beyotime Institute of Biotechnology). Thereafter, the membranes were washed, and the cover slips were mounted using Vectashield mounting medium. Samples were analysed by immunofluorescence microscopy using a confocal laser scanning microscope (Leica, Wetzlar, Germany).
3
Immunohistochemical Analysis of Myocardial Infarction
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The mice were deep anesthetized at the 3rd day after MI or sham surgery. The hearts were quickly removed and fixed in 4% paraformaldehyde, embedded in paraffin and sectioned at 5 microns. Cultured cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.2 % Triton X-100 for 20 min, blocked with 3 % BSA for 60 min at room temperature. Ventricular sections and cells were incubated with primary antibodies against α-actinin (Abcam), F4/80 (Abcam), iNOS (Abcam), connexin43 (Cx43) (Abcam), KCa3.1 (Alomone labs), CD163 (Abcam), CD68 (Abcam) and vimentin (Abcam), and subsequently with fluorescent secondary antibodies Alexa Fluor 488 (Invitrogen), Alexa Fluor 555 (Invitrogen) or Alexa Fluor 647 (Invitrogen). Cells were stained with DAPI for nuclei, and analyzed by confocal microscope (Leica) or scanner (3D Histech), and analyzed by Image-Pro Plus 6.0 software (Media Cybernetics) or Caseviewer software (3D Histech). More than 3 sections of ventricles from different mouse samples were randomly selected to quantify the number of macrophages.
4
Western Blotting Analysis of Cardiac Markers
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Western Blotting was performed as previously described [22] (link). Briefly, the protein samples were mixed with Laemmli sample buffer (1×) and boiled for 5 min. Proteins were subjected to 10% SDS-PAGE and electroblotted onto BioRad, 0.22 µM nitrocellulose membrane (BioRad Laboratories, USA), followed by blocking for 1 h at room temperature using Tris-buffered saline with 0.2% Tween 20 (TBS-T) containing 3% BSA. The membrane was incubated with primary antibodies at 4°C overnight and subsequently incubated with secondary antibodies conjugated with horseradish peroxidase (Sigma Aldrich, USA) for 2 h at RT and washed again with TBS-T. Antibody-reactive proteins were detected by enhanced chemiluminescence using Pierce ECL Plus western blotting detection reagents (Pierce, USA). Primary antibodies for GATA4 (1∶150; Santa Cruz Biotechnology, USA), Connexin43 (CX43; 1∶500; Abcam, USA), Alpha sarcomeric actin (ACTA1; 1∶500; Sigma Aldrich, USA) and Beta Actin (ACTB; 1∶1000; Sigma Aldrich, USA) were used along with goat anti-rabbit and anti-mouse horseradish peroxidase conjugated secondary antibodies (1∶10,000; Sigma Aldrich, USA).
5
Hydrogel Tissue Engineering Protocol
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Gelatin, dopamine, N′, N′-methylene-bisacrylamide (MBA), Poly (ethylene glycol) diacrylate (PEGDA, Mw = 700) tetramethylethylenediamine (TEMED) were all purchased from Sigma (St Louis, USA). Ti2AlC has been purchased from 11 technology Co. LTD (Jilin, China). The Live/Dead cell staining kit was from Molecular Probes (Life Technologies). The Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Japan). The primary antibodies of α-actinin, connexin 43 (CX-43), and von Willebrand Factor (vWF) were acquired from Abcam. F4/80 antibody was purchased from Ebioscience (USA). The alpha-smooth muscle actin (α-SMA) was ordered from Boster Biological Technology (Wuhan, China).
6
Cardiac Cell Culture Protocol
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Poly (ε-caprolactone) (PCL) and gelatin were purchased from Sigma (St Louis, USA). Methanol, chloroform and alcohol were obtained from Macklin Chemical Reagent Company (Shanghai, China). Live/dead cell staining kit, Alexa Fluor 568 donkey anti-rabbit IgG (H&L), and Alexa Fluor 488 donkey anti-mouse IgG (H&L) were from Molecular Probes (Life Technologies). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Japan). The primary antibodies of Ki67, α-actinin, connexin 43 (CX-43), and von Willebrand Factor (vWF) were acquired from Abcam. F4/80 antibody was purchased from Ebioscience (USA). Cell Navigator F-Actin Labeling Kit was from AAT Bioques. Alpha smooth muscle actin (α-SMA) was ordered from Boster Biological Technology (Wuhan, China).
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