Lysates were prepared with radioimmunoprecipitation assay (RIPA) buffer. Protein concentration in the samples was measured using bicinchoninic acid (BCA) protein assay kit and adjusted using water. Samples were loaded onto a 10% acrylamide gel. The gel was run at 120 V for 90 min. Afterward, membrane transfer was performed at 250 mA for 70 min. Membranes were incubated with primary antibodies against NDUFS1 (Thermo Fisher, Cat# PA5-22309), aldolase A (Novus Biologicals, Cat# NBP1-87488), NHE1 (BD Biosciences, Cat# 611775), β-actin (Proteintech, Cat# HRP-60008), and GAPDH (Proteintech, Cat# HRP-60004) used as a loading control; connexin 26 (Thermo Fisher, Cat# 13-5800), connexin 31 (Proteintech, Cat# 12880-1-AP), connexin 43 (Thermo Fisher, Cat# 13-8300), and HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies were applied. The membrane was visualized using ECL.
Connexin 43
Manufactured by Thermo Fisher Scientific
Sourced in United States
Connexin 43 is a protein that forms gap junctions, which are specialized intercellular channels that allow the exchange of ions, small molecules, and electrical signals between adjacent cells. Connexin 43 is widely expressed in various tissues and plays a crucial role in cell-to-cell communication, tissue homeostasis, and coordination of cellular functions.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Proteintech (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Tcs sp2 aobs, by Leica (2 mentions)
Connexin 26, by Thermo Fisher Scientific (1 mentions)
Ndufs1, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Proteintech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Alpha actinin, by Abcam (1 mentions)
Alexa fluor 568, by Abcam (1 mentions)
Alexa fluor 488, by Abcam (1 mentions)
H3k9ac, by Merck Group (1 mentions)
H3k4me3, by Merck Group (1 mentions)
H4 hyper ac, by Merck Group (1 mentions)
Antibodies against gapdh, by Abcam (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Suz12, by Cell Signaling Technology (1 mentions)
Hdac2, by Santa Cruz Biotechnology (1 mentions)
Hdac1, by Santa Cruz Biotechnology (1 mentions)
Dnmt1, by Santa Cruz Biotechnology (1 mentions)
14 protocols using connexin 43
1
Western Blot Analysis of Mitochondrial and Cell Surface Proteins
2
Immunofluorescence Characterization of hiPSC-CMs
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Human iPSC-CMs were fixed in 4% paraformaldehyde for 20 min, followed by washing (PBS) wash. Nonspecific antigenic sites of the fixed cells were blocked with 5% FBS (FBS; Gibco, Gaithersburg, MD, USA) for 1 h at 4 °C. Primary antibodies, including alpha-actinin (Abcam; catalog no. ab68194, Cambridge, UK) and connexin 43 (Thermo Fisher Scientific; catalog no. 71-0700) were hybridized at 4 °C overnight. The samples were then rinsed with PBS and incubated with secondary antibodies: goat anti-mouse IgG (H&L), Alexa Fluor™ 488 (Abcam; catalog no. ab175473), and goat anti-rabbit IgG (H&L), Alexa Fluor 568 (Abcam; catalog no. ab175471), at room temperature for 1 h. All primary and secondary antibodies were prepared in PBS containing 2% BSA (Thermo Fisher Scientific, catalog no. 15260037). Fluorescence images were captured using an inverted fluorescence microscope (OLYMPUS; catalog no. BX51; Tokyo, Japan). The acquired fluorescent images were processed and quantified using ImageJ software.
3
Protein Analysis of Adult Cardiac Cells
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Total proteins from AF-MSCs were isolated as described previously (14 (link)). Adult mouse heart was washed with PBS, homogenized in liquid nitrogen and lysed using 2X SDS lysis buffer. Proteins from cells and mouse heart were fractionated in 7.5~15% gradient SDS/PAGE gel, transferred onto the PVDF membrane and target proteins were detected using antibodies against DNMT1, HDAC1, HDAC2 (Santa Cruz Biotechnology, Texas, USA), SUZ12 (Cell Signaling Technology, MA, USA), BMI1, H3K4me3, H3K9Ac, H4hyperAc (Millipore, MA, USA), EZH2, Connexin43 (Thermo Scientific, IL, USA) and Nkx2.5 (GeneTex, CA, USA). Antibodies against GAPDH (Abcam, Cambridge, UK) were used as a protein loading control. Secondary anti-mouse, anti-rabbit and anti-goat antibodies (DAKO, Glostrup, Denmark), conjugated with horseradish peroxidase, were used and enhanced chemiluminescence was detected using ClarityTM Western ECL Substrate and ChemiDocTM XRS+ system with Image LabTM Software (Bio-Rad Laboratories, CA, USA). Semiquantitative analysis of blots was performed using Image J software (NIH, MD, USA), the relative density of each band was normalized to the GAPDH loading control.
4
Histological Characterization of Cell Sheets
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After detachment cell sheets were frozen in Tissue-Tek (Sakura Finetek, USA) and 7 μm sections were obtained. Cross sections were formalin-fixed and stained by primary antibodies against Connexin43 (cat#71-0700, ThermoFisher Scientific, USA), Ki-67 (cat#ab16667, Abcam, USA), and Gata 4 (cat#PA5-29663, Thermo Fisher Scientific, USA) cleaved caspase-3 (cat#9664S, Cell signaling, USA) for 1 hour. To visualize ECM components cell sheet sections were incubated with a polyclonal antibodies specifically reactive with collagen I (cat#2150-1908, Bio-Rad, USA), collagen 3 (cat#2150-1948, Bio-Rad, USA), and the fibronectin (cat#6328-250, Abcam, USA). After washing the slides were stained for 40 min by the corresponding secondary antibodies (cat#A11001, A11008, A21203, or A21207, all Invitrogen, USA, 1:800, 1h at 37°C).
5
Cardiac Connexin-43 Immunostaining
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Cardiac sections of 20 µm thick were cut in a cryostat at −20 °C. Tissue were incubated with blocking solution with 5% gelatin from cold-water fish skin, 0.5% Triton X-100 and Phosphate Buffer Saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4). Then were incubated with primary rabbit antibody against Connexin-43 (71–0700, THERMOFISHER) at a 1:100 dilution in blocking solution, followed by incubation with the secondary anti-rabbit antibody conjugated with Alexafluor-488 at a 1:1,000 and Alexa Fluor-594 phalloidin at a 1:1,000 diluted in blocking solution. Slices were mounted in Vectashield with DAPI (VECTOR Laboratories). The immunostaining was visualized in a high-resolution fluorescent microscope (LEICA, Germany) and images were acquired at 40×.
6
Western Blotting of Serotonin Receptors
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The performance of western blotting referred to related research [20] . We used rabbit monoclonal antibodies: GAPDH (#ab8245, Abcam, Waltham, MA, USA), 5-HT 1A R (#ab85615, Abcam, MA, USA), 5-HT 1D R (#ab140486, Abcam, MA, USA), 5-HT 1B R (#PA1-41069, Thermo Fisher Scientific, Waltham, MA, USA), 5-HT 1F R (#PA5-106542, Thermo Fisher Scientific, MA, USA), Connexin 43 (#71-0700, Thermo Fisher Scientific, MA, USA), Pannexin 1 (#488000, Thermo Fisher Scientific, MA, USA) and P2Y 1 (#P6487, Sigma-Aldrich, St. Louis, MO, USA). The proteins were then incubated with peroxidaseconjugated goat anti-rabbit IgG (#7074, Cell Signaling Technology, MA, USA). A visualizer was then used to observe the stripe, and the grayscale value was calculated using Image J v1.8.0 (National Institutes of Health, Bethesda, MD, USA).
7
Immunofluorescence Analysis of Cell-Cell Junctions
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Samples were fixed for 20 min with 4% paraformaldehyde and excess paraformaldehyde was removed by washing with Dulbecco’s phosphate-buffered saline (PBS). The samples were permeabilized with 0.3% Triton® X-100 (Nacalai Tesque, Kyoto, Japan), and then incubated with 1% bovine serum albumin (BSA) to block nonspecific binding. Specimens were incubated with primary antibodies against Na+/K+-ATPase (1:300, Upstate Biotechnology, Lake Placid, NY), ZO-1 (1:300, Life Technologies Corp.), and N-cadherin (1:300, BD Biosciences, San Jose, CA), connexin 43 (1:300, Life Technologies Corp.), and vinculin (Merck Millipore, Billerica, MA). Alexa Fluor® 488- or 594- conjugated goat anti-mouse (Life Technologies Corp.) antibodies were used as secondary antibodies at a 1:1000 dilution. Actin staining was performed by incubation with a 1:400 dilution of Alexa Fluor® 488- or 546-conjugated Phalloidin (Life Technologies Corp.). Nuclei were stained with DAPI (Vector Laboratories, Burlingame, CA). The slides were examined with a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Wetzlar, Germany).
8
Immunofluorescence Analysis of Corneal Tissue
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After 14 days of PLGA microsphere injection, rabbits were euthanized, the eye balls were enucleated, and sclerocorneal specimens were prepared using ophthalmic surgical scissors. Samples were fixed in 4% formaldehyde and incubated for 30 minutes in 1% bovine serum albumin to block nonspecific binding. The samples were then incubated overnight at 4°C with antibodies against Na+/K+-ATPase (1 : 300, Upstate Biotechnology, Lake Placid, NY), ZO-1 (1 : 300, Life Technologies Corp.), and connexin 43 (1 : 300, Life Technologies Corp.). Alexa Fluor® 488-conjugated goat anti-mouse (Life Technologies Corp.) was used as a secondary antibody at a 1 : 1000 dilution. Cell morphology was evaluated after actin staining with a 1 : 400 dilution of Alexa Fluor 594-conjugated phalloidin (Life Technologies Corp.). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI, Dojindo Laboratories, Kumamoto, Japan). The samples were examined with a fluorescence microscope (TCS SP2 AOBS; Leica Microsystems, Wetzlar, Germany).
9
Immunofluorescence Imaging of Cell Junctions
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Cells plated in 12-wells plate used for immunofluorescence were cultured under the same conditions and timing as cells on MEAs. Cells were permeabilized after fixation in 4% PFA and blocked before staining with primary antibodies (rabbit anti Connexin 43 (Invitrogen 574366A; 1:200) and mouse anti N-Cadherin (Sigma C2542, 1:100)) and secondary antibodies (Alexa Fluor-488 goat anti-mouse IgG (Life Technology, A21235; 1:250), Alexa Fluor-647 goat anti- rabbit IgG (Life Technology, A11008/A21222; 1:250)). Nuclei were stained with DAPI (Sigma, D9542, 1:40000). Examination was performed by Leica SPE confocal laser scanning and Leica Application Suite Advanced Fluorescence software. Immunofluorescence images were analyzed using Image J version 1.50i (National Institutes of Health, NIH, USA) as described previously[10 (link)].
10
Western Blot Analysis of Protein Levels
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Protein levels were measured by WB with β-actin as the loading control. Protein was extracted from atria samples or cells and quantitated by a BCA assay. Equal amount (10–30 μg) of protein was loaded and separated by SDS-PAGE using 10 or 12% acrylamide gradients, and later transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk and incubated with antibodies against total-AMPK (1:1,000; Cell Signaling Technology/CST, Massachusetts, United States), CD36 (1:1,000; Abcam), collagen Ⅰ (1:1,000; Abcam), collagen Ⅲ (1:1,000; Abcam), connexin-43 (1:200; Invitrogen), CPT1B (1:1,000; Proteintech), GLUT4 (1:500; Proteintech), NFκB (1:1,000; Proteintech), NRF2 (1:1,000; Proteintech), phoso-AMPK (Thr172; 1:1,000; CST), phoso-Akt (Ser473; 1:1,000; CST), phoso-NFκB (1:1,000; CST), PGC1α (1:1,000; Proteintech), SOD2 (1:1,000; Proteintech), TGF-β (1:1,000; Abcam), α-SMA (1:1,000; CST), β-actin (1:5,000; Proteintech). Protein levels were quantified as the intensity of bands using ImageJ software (NIH systems) and standardized to β-actin. Abbreviations are fully illustrated in corresponding figure legends.
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