Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing expression of Cx43 in WT (lane 1) and in Cx43-null astrocytes transfected with full length Cx43 (lane 3), with Cx43 carboxyl terminus (lane 4) and with Cx43M257 (lane 5). Lane 2 corresponds to untransfected Cx43-null cells. Lanes 1–4 correspond to immunoblots performed with Cx43-18A antibody and lane 5 with Cx43-16A. (B) Time courses of calcein FRAP obtained from untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT astrocytes, and from untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares) and Cx43M257 (black circles). Data were obtained from astrocytes cultured from a minimum of 3 litters of mice.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing, Transfection, Cell Culture

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for untreated (black squares) and 48 h carbenoxolone-treated (open triangles) WT and untransfected Cx43-null spinal cord astrocytes exposed to the P2Y1R agonist 2-MeS-ATP. Note that blockade of gap junctional communication in WT astrocytes does not alter the half-maximal response (EC50 value) induced by the P2Y1R agonist. Mean values were obtained from four to seven independent experiments. (B) Dose-response curves obtained for WT (black squares), untransfected (open circles) and transfected Cx43-null astrocytes with full length Cx43 (open squares), Cx43M257 (black circles) and Cx43CT (black triangles). Note that both full length Cx43 and Cx43CT but not Cx43M257 shifted the EC50 values of 2-MeS-ATP obtained for Cx43-null astrocytes to those obtained in WT cells. Data were obtained from 5 to 8 litters.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: Bar histograms showing the mean values of P2Y1R expression levels in WT, untransfected Cx43-null (KO), and in Cx43-null transfected with Cx43 CT, Cx43 truncated at position 257 (M257) and with full length Cx43. An example of western blots for Cx43 and β-actin is shown above.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Expressing, Transfection, Western Blot

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Dose-response curves obtained for WT (black squares) and untransfected (open circles) and Cx43Δ260–280 transfected (black triangles) Cx43-null astrocytes exposed to 2-MeS-ATP, showing that deletion of the Cx43 SH3 domain does not rescue P2Y1 receptor function. Mean±SE values are from 100 to 200 cells obtained from three litters. (B) Dose-response curves obtained from WT (black squares), and Cx43-null astrocytes untreated (open circles) and treated (black triangles) with a membrane permeant peptide corresponding to amino acids 260–280 of Cx43CT. (C) Bar histograms of the mean±SE values of intracellular calcium mobilization induced by 100 nM 2-MeS-ATP recorded from WT, Cx43-null and Cx43-null transfected with Cx43CT and Cx43M257 in the absence and presence of 5 µM PP2. Note that only untransfected and Cx43M257 transfected Cx43-null astrocytes that were not exposed to PP2 did not respond to agonist with intracellular calcium levels similar to WT astrocytes. Mean ± SE values are from 4 litters (**P < 0.001; ANOVA followed by Newman-Keuls’ Multiple Comparison Test).

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Transfection

Journal:

Article Title: Modulation of Astrocyte P2Y 1 Receptors by the Carboxyl Terminal Domain of the Gap Junction Protein Cx43

doi: 10.1002/glia.20598

Figure Lengend Snippet: (A) Western blot showing decreased expression levels of Cx43 following exposure of spinal cord astrocytes to IL-1β (B) Dose-response curves obtained for 2-MeS-ATP performed on Fura-2 loaded WT and Cx43 KO astrocytes treated for 24 h with IL-1β (20 ng/mL). Note that exposure to the cytokine altered the agonist EC50 values in WT astrocytes. About 180 cells from three independent experiments were used in each condition.

Article Snippet: The following primary antibodies were employed: polyclonal antiP2Y1 (1:200; Alomone Labs), and two different polyclonal antiCx43 antibodies (gifts from Dr. E.L. Hertzberg, Albert Einstein College of Medicine), one recognizing Cx43 at position 241–261 (16A) and the other at position 345–360 (18A).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of full length Cx43 and αCT1 peptide. B) αCT1 interaction with ZO-1 PDZ domains as indicated by EDC zero-length cross-linking to GST fusion PDZ1, PDZ2 and PDZ3 polypeptides and neutravidin labeling of biotin-tagged peptide at concentrations of 5, 25 and 50 μM. The deletion of the CT Isoleucine (I) in αCT1-I renders this peptide incompetent to interact with the ZO-1 PDZ2 domain. C) Coomassie blue gel of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT and PKC-ε, with (αCT1) and without (Vehicle) αCT1. The fainter band above GST-Cx43 bands (indicated by lines) in the αCT1 lanes were cut from gels and analyzed by Tandem Mass Spectrometry (MS/MS). The boxes to right of gel show Cx43 CT peptides identified by MS/MS as being cross-linked to αCT1. D) Tandem mass spectrum of a quintuply charged crosslinked peptide (m/z: 674.1) between Cx43 345-366 (a-chain) and αCT1 peptide through Cx43 K346 and E8 in αCT1 (b-chain). Only the b-and y-sequence specific ions are labeled. Arrow indicates ion (b a5 2+ ) consistent with cross-linkage between Cx43 CT lysine K346 and the glutamic acid (E) residue of αCT1 at position −1.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Blots of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT, GST-Cx43 CT QQ/KK in which the lysine (K) residues were mutated to neutral glutamines (Q), PKC-ε and αCT1 (at 5, 10 and 25 μM) and a scrambled αCT1 (M4 scr) variant at the same concentrations. Only αCT1 is seen to be covalently linked by EDC to Cx43 CT in a concentration-dependent manner.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Schematics of Cx43 and the secondary structure of Cx43 CT from amino acid residues Glycine252 (G252) through to Isoleucine 382 (I382). The depiction of secondary structure in 2A has been modified from a diagram originally provided by Sosinsky and co-workers . B) ZDOCK and C) Schrodinger molecular modeling software analysis of the structure of a proposed αCT1-Cx43 CT complex. The protonated structure of αCT1 peptide and Cx43 CT (PDB:1r5s), constrained by a salt-bridge interaction between K346 in the Cx43 CT and the glutamic acid (E) at position −1 of αCT1. The αCT1-Cx43 interaction shown represents that based on the lowest energy minimization score determined in the model. D) Schrodinger molecular modeling software, a 2D map of αCT1-Cx43 CT in anti-parallel orientation showing location of amino acids predicted to bond to each other and the type of bond that is predicted to occur.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Modification, Software

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: SPR was used to analyze interactions of biotin-αCT1 and biotin-αCT1 variant peptides, immobilized to streptavidin-coated chips, with the Cx43 CT (Cx43-CT: amino acids 255 to 382) and Cx43 CT-KK/QQ as analytes, respectively. The mean of three runs is plotted for each analyte concentration. The exposure of the sensor chip to the specific analyte is indicated by the gray area. Sensorgrams obtained for: A) Cx43 CT and biotin-αCT1. B) Cx43 CT-KK/QQ and biotin-αCT1. C) Cx43 CT and biotin-M1 AALAI. D) Cx43 CT-KK/QQ and biotin-M1 AALAI. E) Cx43 CT and biotin-M3 DDLAI. F) Cx43 CT-KK/QQ and biotin-M3 DDLAI.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Variant Assay, Concentration Assay

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Melt curves (top) and first derivative of melt curves (bottom) for ZO-1 PDZ2 at 500 μg/mL in combination αCT1 at concentrations of 25, 50 and 100 μM. B) Temperature maxima (Tm) from Boltzman curves from left-to-right of Cx43 CT (Cx43-CT: amino acids 255 to 382) alone, Cx43 CT in combination with αCT1, and the αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. αCT1, αCT1-I and αCT11 show similar abilities to destabilize (i.e., significantly decrease the Tm of) Cx43 CT. **p<0.01, *** p<0.002, N=6. C) Temperature maxima (Tm) from Boltzman curves from left-to-right of PDZ2 alone, and PDZ2 in combination with αCT1 and αCT1variants including αCT1 variants including: M1 AALAI, M2 AALEI, M3 DDLAI, M4 scrambled, αCT-I and αCT11. M3 DDLAI, αCT1, and αCT11 show similar abilities to stabilize (i.e., significantly increase the Tm of) PDZ2. **p<0.01, ***p<0.002, N=6

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques:

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: A) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with substrate (Cx43-CT: amino acids 255 to 382), but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-tagged αCT1, biotin-tagged αCT1 mutant peptides with alanine substitutions (M1 AALAI, M2 AALEI, M3 DDLAI) and biotin-tagged M4 scrambled. Peptides are at 20 μM. B) Blots of Cx43-pS368 (top) and total Cx43 (bottom) in kinase reactions mixtures including no-kinase controls with Cx43 CT substrate, but no PKC-ε (PKC-minus); Cx43-CT substrate with PKC-ε (PKC-plus); and mixtures containing PKC-ε, Cx43 CT, and biotin-αCT1, biotin-αCT1-I or biotin-αCT11 (RPRPDDLEI with no antennapedia sequence at peptide NT) and biotin-M4 scrambled peptide. Peptides are at 20 μM. C) Chart showing that the ability of unmodified αCT1 and the Cx43 CT interaction-competent peptides biotin-αCT1-I or biotin-αCT11 to induce S368 phosphorylation was 3-5 fold greater than that of non-Cx43 CT interacting peptides. * p<0.05, ** p<0.01, *** p<0.002, N=5 αCT1 and M4, other peptides N=3.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Mutagenesis, Sequencing

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff ischemia-reperfusion (I/R) injury protocols were performed on adult mouse hearts instrumented to monitor LV contractility (protocol in ). LV Systolic responses are shown in 7A-C : (A) Plots of left ventricular (LV) systolic developed pressure against balloon volume ; (B) LV maximal rate of tension development (+dP/dt) against balloon volume; (C) Maximal systolic elastance (E max ) – i.e., the slope from (A) ; (D) Plots of LV end diastolic pressure (EDP) against balloon volume; (E) Maximal rate of relaxation (-dP/dt) against balloon volume; (F) Stiffness, the reciprocal of the slope from (D) ; ( G) Percentage of LV contractile function recovery post-ischemia relative to baseline level. Data shown are mean ± S.E. N=4-8. *p<0.05, ***p<0.001, N=4-8 hearts/group. H) Blots of Cx43-pS368 (top) and total Cx43 (bottom) of LV samples infused with peptide for 20 minutes according to the protocol in . For hearts used in Western blots, the protocol did not proceed to the ischemia and reperfusion phases, being terminated after the peptide infusion step. Only those peptides competent to interact with Cx43 CT increase pS368 levels relative to total Cx43 above vehicle control.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Western Blot

Journal: bioRxiv

Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

doi: 10.1101/668509

Figure Lengend Snippet: Langendorff I/R protocols were performed on adult mouse hearts instrumented to monitor LV contractility. Protocol in , except that a 20-minute peptide infusion was begun after ischemic injury at the initiation of reperfusion. (A) Plots of left ventricular (LV) developed pressure against balloon volume; (B) Maximal systolic elastance (E max ), the slope from (A); (C) Maximal rate of tension development (+dP/dt) against balloon volume; (D) Plots of end diastolic pressure (EDP) against balloon volume; (E) Stiffness, the reciprocal of the slope from (D); (F) Maximal rate of relaxation (-dP/dt) against balloon volume. * p<0.05, *** p<0.001, N=4-8. G) Laser scanning confocal microscopic fields from sections of Vehicle control, αCT1, and αCT11 group hearts stained for Cx43 (green), nuclei (DAPI-blue), and Alexa647-conjugated streptavidin (red). H) Average intensities of biotinylated peptide (indicated by streptavidin Alexa647 fluorescence intensity level relative to background) in Vehicle control, αCT1, and αCT11 groups. ** p<0.05; not significant (ns) N=5 hearts/group. Scale bar = 5 μm.

Article Snippet: Phospho-Connexin43 (Ser368) (Cell Signaling, 3511S, Danvers, MA), anti-Cx43 produced in rabbit (Sigma: C6219, St. Louis, MO), anti-GST produced in goat (GE, 27457701, Little Chalfont, UK).

Techniques: Staining, Fluorescence

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: (A) Top: Representative confocal microscopy images showing Cx43 signal at cell-cell junctions in control wildtype (WT) and PKCε-KO hearts. Bottom: Quantitative confocal microscopy measurements showing amount of Cx43 signal as a percent of total tissue area in WT and PKCε-KO hearts; n for each group indicated in bar. *p<0.001 vs WT. (B) Densitometric measurements from immunoblots of total tissue Cx43 in WT and PKCε-KO hearts under basal conditions. Representative immunoblots are shown below each measurement; n for each group indicated in bar.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy, Control, Western Blot

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: Top: Representative confocal microscopy images showing Cx43 signal at cell-cell junctions in wildtype (WT) and PKCε-KO hearts subjected to 30 min ischemia without (Non-PC) or with preconditioning (PC). Bottom: Quantitative confocal microscopy measurements showing amount of Cx43 signal as a percent of total tissue area. Values are expressed as percent of basal values to account for differences in basal Cx43 signal between WT and PKCε-KO animals; n for each group indicated in bar. *p<0.001 vs. WT non-PC; #p<0.001 vs. PKCε-KO PC.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: (A) Densitometric measurements from immunoblots of total tissue Cx43 in control, non-PC, and PC hearts from WT animals. (B) Densitometric measurements from immunoblots of total tissue Cx43 in control, non-PC, and PC hearts from PKCε-KO animals. Representative immunoblots are shown below each measurement; n for each group indicated in bar.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Western Blot, Control

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: Top: Representative confocal microscopy images showing Cx43 signal at cell-cell junctions in WT hearts perfused for 10 min with a PKCε activator, PKCδ activator, or control peptide before undergoing 30 min of global ischemia. Bottom: Quantitative confocal microscopy measurements showing amount of Cx43 signal as a percent of total tissue area in hearts treated with control peptide (CC), PKCε activator or PKCδ activator; n=3 for each group. *p=0.013 vs. control; #p=0.006 vs. PKCδ activator.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy, Control

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: Densitometric measurements from immunoblots of Cx43 phosphorylated at (A) Ser368, (B) Ser 262, (C) Ser255 and Ser279/282, and (D) Tyr265 in control, non-PC and PC hearts from WT and PKCε-KO animals; n for each group indicated in bar. #p<0.03 vs. WT control; *p<0.001 vs. PKCε-KO control; †p<0.02 vs. WT non-PC; §p=0.003 vs. WT PC. Representative immunoblots are shown below each measurement.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Western Blot, Control

Journal:

Article Title: Protein Kinase C? Mediates Salutary Effects on Electrical Coupling Induced by Ischemic Preconditioning

doi: 10.1016/j.hrthm.2007.05.030

Figure Lengend Snippet: (A) Representative confocal microscopy images showing amount of Cx43 phosphorylated at Ser368 in cell-cell junctions. (B) Quantitative confocal microscopy measurements showing amount of phosphorylated at Ser368 in cell-cell junctions expressed as a proportion of total Cx43 in junctions; n for each group indicated in bar. *p=0.005 vs. WT control; #p=0.015 vs. WT PC; §p=0.049 vs. PKCε-KO control.

Article Snippet: Antibodies used in this study included a rabbit polyclonal antibody (Zymed) directed against epitopes in the C-terminus of rat Cx43 (immunoblotting, 1:5000 dilution); a mouse monoclonal anti-Cx43 antibody (Chemicon MAB3068) (immunohistochemistry and confocal microscopy, 1:400 dilution); a rabbit polyclonal anti-phospho-Cx43 antibody directed against Ser368 (Cell Signaling) (immunoblotting, 1:500 dilution; immunostaining,1:100 dilution); rabbit polyclonal anti-phospho-Cx43 antibodies directed against Ser262, Ser279/282, Ser255 or Tyr265 (Santa Cruz) (immunoblotting); a monoclonal anti-PKCε antibody (BD Biosciences) (immunoblotting, 1:250 dilution); a polyclonal anti-PKCδ antibody (Santa Cruz) (immunoblotting, 1:750 dilution); a monoclonal anti-GAPDH antibody (RDI) (immunoblotting, 1:5000 dilution), and a polyclonal anti-actin antibody (Santa Cruz) (immunoblotting, 1:1000 dilution).

Techniques: Confocal Microscopy, Control

Journal: Korean Circulation Journal

Article Title: Preventive Effects of the Angiotensin-II Receptor Blocker on Atrial Remodeling in an Ischemic Heart Failure Model of Rats

doi: 10.4070/kcj.2013.43.10.686

Figure Lengend Snippet: Even distribution of connexin 43 protein on the intercalated disc was observed in the sham group (A: magnification: ×400). Connexin 43 protein stain was not observed on the intercalated disc in the heart failure group (B: magnification: ×400). A slight connexin 43 protein stain was observed on the intercalated disc in the heart failure-ARB group (C: magnification: ×400). Western blot of connexin 43 protein showed a lower expression in the heart failure-ARB group (D). ARB: angiotensin-II receptor blocker.

Article Snippet: The immunohistochemical stain for connexin 43 (rabbit polyclonal anti-Cx43 antibody, 1/100, Zymed-laboratories, CliniSciences, France) showed a significant amount of plasmalemmal distribution of connexin 43 in the heart failure group.

Techniques: Staining, Western Blot, Expressing