Protein was extracted from myometrial tissues and cells using the RIPA lysis buffer. It is noteworthy that cell lysis ought to occur within 5 min of being removed from a hypoxic environment. Protein concentration was measured using a BCA assay kit (#23227, Thermo Scientific, USA), according to the manufacturer’s instructions. Western blot protein samples were loaded in SDS-PAGE gel, separated by electrophoresis, and transferred onto PVDF (polyvinylidene fluoride) membranes (IPVH00010, Millipore, Darmstadt, Germany). Protein levels were quantified by a ChemiDoc XRS+. β-Actin was used as a loading control. The western blotting antibodies used were anti-oxytocin receptor (1:5000, ab181077, Abcam), anti-Connexin43 (1:2000, #3512, Cell Signaling Technology), anti-HIF-1α (1:1000, ab2185, Abcam), anti-HIF-2α (1:1000, ab199, Abcam), COX-2 (1:2000, ab179800, Abcam), and anti-β-actin (1:1000, ab8226, Abcam). Every experiment was replicated at least three times.
Anti connexin 43
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Anti-connexin 43 is a primary antibody that binds specifically to the connexin 43 (Cx43) protein. Connexin 43 is a gap junction protein that plays a critical role in cell-to-cell communication.
Automatically generated - may contain errors
Lab products found in correlation
Ab181077, by Abcam (2 mentions)
Anti total creb, by Cell Signaling Technology (2 mentions)
Anti phospho creb, by Cell Signaling Technology (2 mentions)
Ipvh00010, by Merck Group (1 mentions)
Ab2185, by Abcam (1 mentions)
Ab199, by Abcam (1 mentions)
Ab179800, by Abcam (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Ab8226, by Abcam (1 mentions)
Wheat germ agglutinin (wga), by Thermo Fisher Scientific (1 mentions)
Anti α smooth muscle actin, by Abcam (1 mentions)
Anti α sarcomeric actin, by Merck Group (1 mentions)
Anti cd31, by Abcam (1 mentions)
Ab7817, by Abcam (1 mentions)
Ab62557, by Abcam (1 mentions)
L7543, by Merck Group (1 mentions)
Af7018, by Affinity Biosciences (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab150116, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
7 protocols using anti connexin 43
1
Myometrial Protein Extraction and Analysis
2
Immunostaining of Cardiac Markers
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Immunostaining was performed with the primary antibodies including anti–α-sarcomeric actin (Sigma Aldrich, MO, US); anti–α-smooth muscle actin (Abcam, Cambridge, United Kingdom); anti-CD31 (Abcam, Cambridge, United Kingdom); anti-connexin43 (Cell Signaling Technology, MA, USA); wheat germ agglutinin (Thermo Fisher Scientific, Waltham, USA). DAPI was used for nuclear counterstaining.
3
Immunofluorescence Analysis of Cellular Markers
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Anti-α-SMA (ab7817, Abcam, Cambridge, UK), anti-GORASP2 (ab204335, Abcam, Cambridge, UK), anti-LC3b (L7543, Sigma-Aldrich, St. Louis, MO, USA), Alexa 594 goat anti-mouse secondary antibody (ab150116, Abcam, Cambridge, UK) or Alexa goat anti-rabbit 488-IgG (ab150077, Abcam, Cambridge, UK), anti-oxytocin receptor (ab181077, Abcam, Cambridge, UK), anti-Connexin 43 (#3512, Cell Signaling Technology, Beverly, MA, USA), anti-Beclin 1 (ab62557, Abcam, Cambridge, UK) and anti-β-actin (AF7018, Affinity, San Francisco, CO, USA).
4
Western Blot Analysis of Hepatic Proteins
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Protein extracts of liver tissues were loaded on sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (EMD Millipore, Burlington, MA, USA) after electrophoresis. Protein concentration was determined with a bicinchoninic acid protein assay kit (Beyotime Biotechnology, Shanghai, China). The proteins were marked with anti-Connexin 43 (Cell Signaling Technology; Danvers, Massachusetts, USA, #3512S, 1:1,000), N-cadherin (Cell Signaling Technology; Danvers, Massachusetts, USA, #13116S, 1:1,000), GAPDH (Cell Signaling Technology; Danvers, Massachusetts, USA, #2118s, 1:1,000), TOM20 (Proteintech; Wuhan, China, #11802-1-AP, 1:1,000), or collagen I (Abcam; Cambridge, CB2 0AX, UK, #ab117119,1:1,000). Enhanced chemiluminescence was used for final detection. Secondary antibodies were used at a dilution of 1:10,000. Gels were imaged using a Bio-Spectrum Gel Imaging System (Bio-Rad Laboratories, Hercules, CA, USA), and band intensity was measured with Photoshop CS6 software (Adobe, San Jose, CA, USA). Cx43 protein bands were normalized to TOM2. Mitochondria from LX-2 cells were collected with a mitochondria isolation kit (Beyotime Biotechnology, Shanghai, China).
5
Immunofluorescence Staining of Mouse Cardiac Cryosections
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Short axis cryosections of fresh unfixed mouse hearts were prepared by the UCLA Translational Pathology Core Laboratory. Following thawing and drying of sections, they were fixed with 4% PFA in PBS for 1 min. Sections were then blocked in 10% Goat serum in PBS for 1 h at room temperature, incubated with primary antibodies for 1 h at room temperature, and incubated with secondary antibodies for 30 min at room temperature. Antibodies used included anti-α-actinin (1:1000, clone EA-53, #A7732, Sigma-Aldrich), anti-connexin 43 (1:100, Cell Signaling #3512, Danvers, MA, USA), anti-N-cadherin (1:100, Developmental Studies Hybridoma Bank #MNCD2, Iowa City, IA, USA), anti-mouse IgG AlexaFluor 555 (1:1000, ThermoFisher #A31570), anti-rat IgG AlexaFluor 488 (1:1000, ThermoFisher #A21208), and anti-rabbit IgG AlexaFluor 555 (1:1000, ThermoFisher #A21428).
6
Immunostaining of Stem Cell Markers
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OC explants or cells were fixed with 4% paraformaldehyde (Biosesang, Gyeonggi-do, Korea) for 15 min at room temperature and permeabilized using 0.2% Triton-X 100 in PBS (PBST). After blocking with 1% bovine serum albumin in 1× PBST, samples were incubated with primary antibodies at 4 °C for 24 h. The primary antibodies were anti-phospho-CREB (#9198, Cell Signaling), anti-total-CREB (#9197, Cell Signaling), anti-connexin 43 (#3512, Cell Signaling), and anti-SOX2 (1:500, Cell Signaling; #4900). Samples were washed thoroughly and incubated with secondary antibodies tagged with fluorescein isothiocyanate (FITC) or cyanine 3 (Cy3; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. F-actin was stained using phalloidin–Texas red (Invitrogen, Molecular Probes, Carlsbad, CA, USA), and nuclei were counterstained with 4′6,-diamidino-2-phenylindole (DAPI; Invitrogen). Coverslips were mounted onto slides with mounting medium (Vector Laboratories, Burlingame, CA, USA). The immunostained cells were observed using a Zeiss LSM 700 confocal microscope (Carl Zeiss Meditec, Jena, Germany).
7
Western Blot Analysis of Signaling Proteins
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Protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), and blocked with 5% skim milk in PBS containing 0.05% Tween 20 (PBST) for 1 h. The membranes were incubated with primary antibodies at 4 °C overnight. After washing with PBST, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (GenDepot, Katy, TX, USA) for 1 h at room temperature. Following washing of the membrane, the immunoblot bands were visualized using ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc, Grand Island, NY, USA). The following antibodies were used: anti-phospho-PKA substrate (#9624, Cell Signaling, Danvers, MA, USA), anti-cyclin D1 (#55506, Cell Signaling), anti-phospho-CREB (#9198, Cell Signaling), anti-total-CREB (#9197, Cell Signaling), anti-connexin 43 (#3512, Cell Signaling), anti-cleaved caspase-3 (#9661, Cell Signaling), and anti-cleaved PAPR (ab32064, Abcam, Cambridge, UK).
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