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: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative western blots suggest that neither total connexin43 (Cx43) nor Cx43 phosphorylated at serine-368 (pCx43-S368) significantly change in response to acute exposure to osmotic agents. Lane 7 contains a positive control using hearts exposed to 60 min of room temperature ischemia, a condition shown to upregulate Cx43 pS368. B. Summary data plotted with mean and standard deviation error bars do not reveal significant differences in either total or pCx43-S368 for all conditions (N=3 hearts per condition. p=n.s. via ordinary one-way ANOVA applying Dunnett’s correction). C. Representative fluorescent images of calcein-AM fluorescence in control cultures (N=13 plates, n=220 cells) and cultures exposed to mannitol (N=7 plates, n=116 cells), albumin (N=4 plates, n=77 cells), Dextran 70kDa (N=4 plates, n=102 cells), Dextran 2MDa (N=4 plates, n=95 cells), and carbenoxolone (CBX, N=6 plates, n=110 cells). Measurements were made before bleaching (Pre-bleach), just after bleaching (t=0s), halfway through and at the end of the recovery period (t=150 and 300s, respectively). D. Fluorescence recovery after photobleaching (FRAP) plot of bleached cells during 300s post bleaching. Solid lines represent averages at each point, and shaded areas represent the standard error of the mean. E. Normalized recovery demonstrates that only CBX significantly decreased FRAP relative to control. *p<0.05, nested ANOVA applying Dunnett’s correction with culture plate nested under treatment type.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Western Blot, Positive Control, Standard Deviation, Fluorescence

Journal: bioRxiv

Article Title: EXTRACELLULAR PERINEXAL SEPARATION IS A PRINCIPAL DETERMINANT OF CARDIAC CONDUCTION

doi: 10.1101/2023.05.25.542366

Figure Lengend Snippet: A. Representative images of ventricular tissue slices perfused with 0.5, 3, and 10kDa fluorescent dyes. Panels demonstrate extracellular diffusion; Cx43 signal is used to identify the intercalated disc (ID), and nuclei are shown alone and with overlay of all signals. B. A region of interest is identified around the Cx43 ID signal and intensity of signal is averaged to identify the peak of the Cx43 signal. The same spatial averaging with 0.5, 3, and 10kDa signal demonstrates a dip in 3 and 10kDa signal with a peak in the 0.5kDa dye demonstrating the 0.5kDa dye permeates the ID, but the other two dyes do not. C. The intensity of the dye fluorescent signal at the peak of the Cx43 signal is normalized to the intensity of signal beyond the ID to reveal that 3 (n=3 images) and 10kDa (n=4 images) fluorescent signal are significantly lower than 0.5kDa (n=7 images) signal intensity. *p<0.05 relative to 0.5kDa, one way ANOVA with Dunnett’s correction.

Article Snippet: Electrophoresis was performed to separate proteins which were then transferred to a PVDF membrane, blocked with 5% bovine serum albumin for 1 hour at room temperature and incubated overnight with a primary antibody against the principal ventricular GJ protein Cx43 phosphorylated at Ser368 (pCx43, 1:1000, #3511S, Cell Signaling Technologies), at 4°C.

Techniques: Diffusion-based Assay

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: Stem Cells International

Article Title: Inhibition of G9a Histone Methyltransferase Converts Bone Marrow Mesenchymal Stem Cells to Cardiac Competent Progenitors

doi: 10.1155/2015/270428

Figure Lengend Snippet: Shared expression of progenitor markers in atrial and bone marrow-derived PBCs. PBCs were collected from either (a–e) atrial cultures or (f–j) BIX01294 plus CCM-treated MSCs and immunostained following (a–c; e–h, i) their attachment on Cell-Tak coated chamber slides or (d, i) being cytospun onto glass slides. Subsequent immunofluorescent labeling of the cells (shown in gray) was used to detect the expression of (a, f) Sca-1 (b, g) CD90 (c, h) Isl-1, (d, i) Cx43, and (e, j) Cx40. Cultures were counterstained with DAPI to visualize nuclei (blue). Scale bar = 25 μ m.

Article Snippet: Staining with anti-muscle α -actinin (EA-53, Sigma), desmin (D8281, Sigma), titin (9D10, DSHB), connexin40 (Cx40, 36-5000, Life Technologies), Cx43 (13-8300, Life Technologies), CD90 (553016, BD Pharmingen) and Sca-1 (16-598, eBioscience) antibodies followed methanol or paraformaldehyde fixation for 10 minutes, multiple PBS washes, and overnight incubation with 5% BSA/PBS blocking solution.

Techniques: Expressing, Derivative Assay, Labeling

Journal: Cancers

Article Title: LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

doi: 10.3390/cancers14092303

Figure Lengend Snippet: Characterization of murine glioma cell lines (GL261, CT2A and ALTS1C1) following LDH-A shRNA knock-down . LDH-A and LDH-B mRNA levels by ddPCR ( A , B ); protein expression on Western blot analyses ( C – E ); and LDH enzyme activity ( F ) in control NC and LDH-A KD cell lines (GL261, CT2A, ALTS1C1). LDH-B/LDH-A mRNA expression ratios ( G ) and LDH-B/LDH-A Western blot ratios ( H ) for control NC and LDH-A KD cell lines (GL261, CT2A, ALTS1C1). n = 3, ±SEM. The native Western blot for Panel E is shown in the .

Article Snippet: The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako).

Techniques: shRNA, Knockdown, Expressing, Western Blot, Activity Assay, Control

Journal: Cancers

Article Title: LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

doi: 10.3390/cancers14092303

Figure Lengend Snippet: Metabolic energy generation of NC and LDH-A KD murine glioma cell lines . Glycolytic proton efflux rate (glycoPER): basal ( A ) and compensatory glycolysis ( B ) assessed by Seahorse XF analyzer (30,000 of NC and LDH-A KD GL261, CT2A, ALTC1S1 cells were seeded 4 h before the experiments). Results were normalized per 10,000 cells (n = 6, mean ± SEM); Rot/AA, rotenone + antimycin A; 2-DG, 2-Deoxy-D-glucose). Energy map of six tested cell lines charting mitochondrial ATP (mito ATP) versus glycolysis-generated ATP (glycol ATP) production; mean ± SD ( C ). Representative profiles of real-time glycoPER for GL261 ( D ), CT2A ( E ) and ALTS1C1 ( F ) cell lines, comparing NC and LDH-A KD. Representative profiles of a real-time OCR profile for GL261 ( G ), CT2A ( H ) and ALTS1C1 ( I ) cell lines, comparing NC and LDH-A KD. Values are mean, ±SEM; n = 5 (GL261); 4 (CT2A); 7 (ALTS1C1).

Article Snippet: The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako).

Techniques: Generated

Journal: Cancers

Article Title: LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

doi: 10.3390/cancers14092303

Figure Lengend Snippet: Overexpression of genes involved in oxidative phosphorylation in GL261 LDH-A KD cells. Transcripts per million (TPM) expression values were plotted in individual cell lines for genes directly involved in lactate metabolism and export (LDH-A, LDH-B, and SLC16A3) ( A ). Gene Set Enrichment Analysis (GSEA) for a single pathway (GO_OXIDATIVE_ PHOSPHORYLATION) is shown for each GBM cell line. The analysis shows the enrichment of this pathway in LDH-A-depleted vs. control cells ( B ). TPM values for enzymes involved in oxidative phosphorylation ( C ). The Z-transformed scores of individual genes within the oxidative phosphorylation (GO) pathway were plotted across each cell line (GL261, CT2A and ALTS1C1). The experiment was performed in triplicate, with rows representing each sample and columns representing individual genes ( D ). Expression (TPM) of pyruvate dehydrogenase alpha 1 (PDHA1) and aconitase 1 (ACO1) was plotted ( E ). PDHA1 is a nuclear-encoded mitochondrial matrix multienzyme complex that provides the primary link between glycolysis and the tricarboxylic acid (TCA) cycle by catalyzing the irreversible conversion of pyruvate into acetyl-CoA; ACO1 is a bifunctional, cytosolic protein that functions as an essential enzyme in the TCA cycle. Significant differences are indicated by: * p < 0.05, ** p < 0.01, and *** p < 0.001. provides the semi-raw data from the RNASeq analysis that was used to generate the figures.

Article Snippet: The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako).

Techniques: Over Expression, Phospho-proteomics, Expressing, Control, Transformation Assay

Journal: Cancers

Article Title: LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

doi: 10.3390/cancers14092303

Figure Lengend Snippet: The effect of LDH-A knock-down on cells in vitro and s.c. tumor growth, in vivo in immune-competent mice . Growth profiles and doubling times of GL261, ALTS1C1 and CT2A cells in vitro (Panels A – D ) (mean ± SEM) and tumors in C57BL/6 mice (Panels E – H ) with and without LDH-A shRNA knock-down (mean, ± SD). Note that the doubling times for GL261 NC tumors (Panel E) were estimated after the initial delay in tumor growth (0~40 days).

Article Snippet: The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako).

Techniques: Knockdown, In Vitro, In Vivo, shRNA

Journal: Cancers

Article Title: LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

doi: 10.3390/cancers14092303

Figure Lengend Snippet: The effect of LDH-A knock-down on s.c. tumor growth in nude mice . Growth profiles of GL261, ALTS1C1 and CT2A tumors (Panels A – C ), and estimated tumor doubling times (Panel D ). Mean, ± SD. Comparison between tumor doubling times in nude mice and C57BL/6 mice (Panel E ).

Article Snippet: The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako).

Techniques: Knockdown, Comparison

Journal: Cancers

Article Title: LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

doi: 10.3390/cancers14092303

Figure Lengend Snippet: Native-polyacrylamide gel electrophoresis LDH zymograms for ex vivo tissue and s.c. GL261, CT2A and ALTS1C2 tumors. Electrophoretic patterns in the heart and skeletal muscle as well as s.c. tumors from NC and LDH-A KD GL261, CT2A and ALTS1C1 tumors (Panel A ), corresponding LDH isoform profiles (Panel B ); n = five independent studies.

Article Snippet: The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako).

Techniques: Polyacrylamide Gel Electrophoresis, Ex Vivo

Journal: Cancers

Article Title: LDH-A—Modulation and the Variability of LDH Isoenzyme Profiles in Murine Gliomas: A Link with Metabolic and Growth Responses

doi: 10.3390/cancers14092303

Figure Lengend Snippet: H&E and IHC staining for LDH-A and LDH-B protein expression in s.c. GL261 and CT2A tumors—both LDH-A KD and NC controls . H&E staining for GL261 NC and LDH-A KD tumors ( Aa ); LDH-A staining ( Ab ) and LDH-B staining ( Ac ). GL261 NC and LDH-A KD tumors were grown in immunocompromised nude mice. Quantification of percentage LDH-A tumor expression ( B ) and percentage LDH-B tumor expression ( C ); ±SEM. A similar presentation is shown for CT2A tumors, grown in immune-competent C57BL/6 mice (Panels D – F ).

Article Snippet: The small 5-day tumors from GL261 NC and KD were stained with immune markers: anti-CD68 antibody (Catalog No. TA1518, Boster), anti-CD4 (Catalog No. AF554, R & D Systems) and anti-CD3 antibody (Catalog No. A0452, Dako).

Techniques: Immunohistochemistry, Expressing, Staining