polyclonal nav1 5 antibody  (Alomone Labs)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Toward Personalized Medicine: Using Cardiomyocytes Differentiated From Urine-Derived Pluripotent Stem Cells to Recapitulate Electrophysiological Characteristics of Type 2 Long QT Syndrome

doi: 10.1161/JAHA.115.002159

Figure Lengend Snippet: Human UhiPS cells differentiated into functional cardiomyocytes. A, Transcriptional profile of control hiPS cells derived from skin fibroblasts (control FhiPS-CMs) and from urine cells (control UhiPS-CMs and A561P-UhiPS CMs) at days 5, 18, and 28 of cardiac differentiation. Quantitative reverse transcription polymerase chain reaction analysis was performed on pluripotent stem cell markers ( OCT3/4 , NANOG , SOX2 ), on cardiomyocyte markers ( NKX2-5 , GJA1 , GJA5 , RYR2 ), and on key genes encoding cardiac ion channels ( SCN5A , CACNA1C , CACNA1G , KCND3 , KCNQ1 , KCNH2 and KCNJ2 ) to show enrichment for the cardiomyocyte population. The expression for each gene and sample (n=5 per condition) was normalized to the ß-actin gene ACTB and was calculated relative to the median expression level. Raw minimum and maximum values were taken as a reference for heat map representation. B, (top) In control UhiPS-CMs, representative immunofluorescence images of the cardiac sarcomeric protein α-actinin (green, left) and costaining of myosin light chain 2a (MLC2a; green, middle) and myosin light chain 2v (MLC2v; red, middle) and troponin I and connexin 43 (green and red, respectively, right); (bottom) in A561P-UhiPS CMs, representative immunofluorescence images of α-actinin (red, left), costaining of MLC2a (red; middle) and MLC2v (green, middle) and troponin I and connexin 43 (green and red, respectively, right). Scale=5 μm. CM indicates cardiomyocytes; FhiPS, foreskin fibroblast–derived human induced pluripotent stem cells; max, maximum; min, minimum; UhiPS, urine-derived human induced pluripotent stem cells.

Article Snippet: For type 2 cells, after methanol fixation, cells were permeabilized with 0.1% Triton X-100, blocked with 5% PBS-BSA and stained with primary antibodies against α-actinin (Abcam), troponin I (Santa Cruz Biotechnology), MLC2a (Abcam), MLC2v (Proteintech Europe), connexin 43 (Chemicon), HERG (Santa Cruz Biotechnology), or Nav1.5 (Alomone Labs).

Techniques: Functional Assay, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: SNTA1 Gene Rescues Ion Channel Function in Cardiomyocytes Derived from Induced Pluripotent Stem Cells Reprogrammed from Muscular Dystrophy Patients with Arrhythmias

doi: 10.1101/2022.01.25.477696

Figure Lengend Snippet: NaV1.5 protein level is significantly reduced in patient-specific DMD iPSC-CMs. (a) Representative Western blot for each antibody used. The bands within the blue rectangles at ∼250 KDa and below 50 KDa correspond to NaV1.5 and Kir2.1, respectively. About 50K cells were collected to quantify total NaV1.5, Kir2.1 and actinin levels in control, heterozygous and hemizygous DMD cells. (b) Scatter plots of NaV1.5 and Kir2.1 detected in control, female and DMD iPSC-CMs. NaV1.5 and Kir2.1 protein levels were normalized to actinin (loading control). (c) Representative Western blot after biotinylation and protein precipitation with streptavidin magnetic beads. (d) Scatter plots of biotinylated NaV1.5 and Kir2.1 from control, female , and DMD iPSC-CMs. Fifty μg of biotinylated protein was loaded. Errors bars represent SEM. The n -values are in parentheses. Two-tailed Mann-Whitney test. *** P

Article Snippet: Primary antibodies were used at different dilutions in block solution: Troponin I (#MAB1691, Millipore) was used at 1:500, Kir2.1 (#APC-026, Alomone) antibody was used at 1:200, Nav1.5 (#AGP-008, Alomone) was used at 1:200, Dystrophin MANDRA1 (#D8043, Sigma) was used at 1:100, and Phalloidin 488 (#A12379, Invitrogen) at 1:500 (it comes with a fluorophore conjugated so no secondary Ab incubation was needed, stains F-actin).

Techniques: Western Blot, Magnetic Beads, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: SNTA1 Gene Rescues Ion Channel Function in Cardiomyocytes Derived from Induced Pluripotent Stem Cells Reprogrammed from Muscular Dystrophy Patients with Arrhythmias

doi: 10.1101/2022.01.25.477696

Figure Lengend Snippet: Electrophysiological analysis in Control 2 (human foreskin-derived BJ iPSC-CMs). ( a ) Representative AP trace of ventricular-like control BJ iPSC-CMs obtained at 1 Hz of pacing. Inset . First derivative with respect to time (dV/dt). ( b–f) Action potential properties. Recordings at 1 and 2 Hz were similar to those obtained from the healthy donor patient derived-iPSC-CMs (Control 1). ( g-i) Current traces, I/V curves, and normalized current densities for NaV1.5, CaV1.2, and Kir2.1 ion channels, respectively. Data obtained from the control BJ iPSC-CMs (Control 2) were similar to the other control iPSC-CMs.

Article Snippet: Primary antibodies were used at different dilutions in block solution: Troponin I (#MAB1691, Millipore) was used at 1:500, Kir2.1 (#APC-026, Alomone) antibody was used at 1:200, Nav1.5 (#AGP-008, Alomone) was used at 1:200, Dystrophin MANDRA1 (#D8043, Sigma) was used at 1:100, and Phalloidin 488 (#A12379, Invitrogen) at 1:500 (it comes with a fluorophore conjugated so no secondary Ab incubation was needed, stains F-actin).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: SNTA1 Gene Rescues Ion Channel Function in Cardiomyocytes Derived from Induced Pluripotent Stem Cells Reprogrammed from Muscular Dystrophy Patients with Arrhythmias

doi: 10.1101/2022.01.25.477696

Figure Lengend Snippet: SCN5A , KCNJ2 and CACNA1C mRNA expression in control, DMD, and female iPSC-CMs. (a) SCN5A mRNA expression was increased in iPSC-CMs from hemizygous and heterozygous DMD individuals (top), as well as in the human left ventricle heart tissue from a Becker MD individual compared to a healthy subject (bottom). (b) KCNJ2 mRNA levels were higher in both hemizygous and heterozygous iPSC-CMs (top), like those found in human left ventricle heart tissue from Becker DM patients (bottom) when compared to the corresponding control. (c) CACNA1C mRNA expression was not significant different among tested groups from either iPSC-CMs (top) or left ventricle tissues (bottom). mRNA levels were determined by qRT-PCR and calculated by the comparative Ct method (2 -ddCt ) normalized to the internal control 18s rRNA. Errors bars represent SEM. The n -values are in parentheses. Two-tailed Mann-Whitney test. ** P

Article Snippet: Primary antibodies were used at different dilutions in block solution: Troponin I (#MAB1691, Millipore) was used at 1:500, Kir2.1 (#APC-026, Alomone) antibody was used at 1:200, Nav1.5 (#AGP-008, Alomone) was used at 1:200, Dystrophin MANDRA1 (#D8043, Sigma) was used at 1:100, and Phalloidin 488 (#A12379, Invitrogen) at 1:500 (it comes with a fluorophore conjugated so no secondary Ab incubation was needed, stains F-actin).

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY

Journal: Pharmaceuticals

Article Title: Synergistic Adverse Effects of Azithromycin and Hydroxychloroquine on Human Cardiomyocytes at a Clinically Relevant Treatment Duration

doi: 10.3390/ph15020220

Figure Lengend Snippet: Nav1.5 and Cx43 protein expression in iPSC-CMs treated with HCQ and/or AZM. ( A ) Two representative Western blots showing the expression of Nav1.5 and Cx43 in iPSC-CMs under different drug treatment conditions. ( B ) Quantitation of protein expression levels of Nav1.5 in iPSC-CMs under different conditions; N = 6 independent differentiations. ( C ) Quantitation of protein expression of Cx43 in iPSC-CMs under different drug treatment conditions; N = 6 independent differentiations. ( D ) Representative images showing immunostaining for Nav1.5 (green) and Cx43 (red) in iPSC-CMs under different drug treatment conditions. Cell nuclei are shown in blue (Hoechst). Statistical evaluation was performed using one-way ANOVA with Tukey’s multiple comparison test (** p

Article Snippet: Membranes were blocked in 5% milk in TBS-T for 30–45 min at room temperature and probed with anti-Cx43 (clone 4E6.2; 1:1000; mouse monoclonal, Merck Millipore, Darmstadt, Germany, MAB3067), anti-Nav1.5 (1:200; rabbit polyclonal, Alomone Labs, Jerusalem, Israel, ASC-005), or anti-EEF2 (1:5000; rabbit polyclonal, Abcam, Cambridge, UK, ab40812) antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibodies goat anti-mouse (1:10,000; Sigma Aldrich, St. Louis, MO, USA, A2304) or goat anti-rabbit (1:10,000; Cell Signaling, Danvers, MA, USA, 7074S) for 1 h at room temperature.

Techniques: Expressing, Western Blot, Quantitation Assay, Immunostaining

Journal: Basic Research in Cardiology

Article Title: The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

doi: 10.1007/s00395-009-0012-8

Figure Lengend Snippet: a Overview ( left panel ) and detail ( right panel ) of embryonic mouse heart (ED14.5) indicating low Na v 1.5 labeling intensity in ventricular subepicardium ( asterisks ) and abundant Na v 1.5 in the trabeculated subendocardium. b In situ hybridization of ventricular myocardium in embryonic heart (ED14.5; overview and magnified boxed section in the right panel ) displaying a transmural gradient in Scn5a mRNA expression with lower expression in the subepicardium ( asterisks ) compared to the trabeculated subendocardium ( arrows ). For comparison, the homeodomain transcription factor Irx5 shows a similar transmural distribution pattern ( lv left ventricle, rv right ventricle, ivs intraventricular septum)

Article Snippet: Antibodies The following primary antibodies were used: rabbit polyclonal anti-Nav1.5 (1:200, Alomone Laboratories, ASC-005), mouse monoclonal anti-alpha-actinin (1:1000, Sigma), mouse monoclonal anti-desmin (1:200, Monosan, MON-3001), mouse monoclonal anti-Cx43 (1:200, BD Biosciences, 610061), rabbit polyclonal anti-Cx40 (1:250, Chemicon), rabbit polyclonal anti-HCN4 (1:200, Chemicon, AB5808), and rabbit polyclonal anti-cardiac troponin I (anti-cTnI, 1:1000, HytestLtd).

Techniques: Labeling, In Situ Hybridization, Expressing

Journal: Basic Research in Cardiology

Article Title: The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

doi: 10.1007/s00395-009-0012-8

Figure Lengend Snippet: a Immunohistochemical analysis of cryosections from adult murine heart showing a detailed view of the sinoatrial node ( SAN ; marked region ). The SAN is clearly demarcated by the presence of HCN4 staining and the absence of Cx43 labeling ( middle panel ) and displays low levels of Na v 1.5 staining. In contrast, abundant Na v 1.5 staining is visible in the surrounding right atrial ( ra ) tissue. b In situ hybridization of embryonic heart (ED14.5) showing mRNA expression of Scn5a , CtnI and Cx43 ( in blue ). The CtnI -positive and Cx43 -negative SAN (denoted by arrow ) shows low Scn5a mRNA expression ( pv pulmonary vein, rscv right superior caval vein). c Immunohistochemical analysis of cryosections from adult murine heart showing a detailed view of the atrioventricular node ( AVN ; marked region ). The AVN shows intense staining for HCN4, but low expression of both Cx40 and Na v 1.5, whereas clear Na v 1.5 staining is visible in the atria ( a ) and working myocardium ( w )

Article Snippet: Antibodies The following primary antibodies were used: rabbit polyclonal anti-Nav1.5 (1:200, Alomone Laboratories, ASC-005), mouse monoclonal anti-alpha-actinin (1:1000, Sigma), mouse monoclonal anti-desmin (1:200, Monosan, MON-3001), mouse monoclonal anti-Cx43 (1:200, BD Biosciences, 610061), rabbit polyclonal anti-Cx40 (1:250, Chemicon), rabbit polyclonal anti-HCN4 (1:200, Chemicon, AB5808), and rabbit polyclonal anti-cardiac troponin I (anti-cTnI, 1:1000, HytestLtd).

Techniques: Immunohistochemistry, Staining, Labeling, In Situ Hybridization, Expressing

Journal: Basic Research in Cardiology

Article Title: The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

doi: 10.1007/s00395-009-0012-8

Figure Lengend Snippet: Distribution of Na v 1.5 in the atrioventricular bundle ( AVB ) and His-Purkinje system in adult murine myocardium. a The AVB lacks Cx43 expression, is HCN4-positive, and shows a high Na v 1.5 labeling intensity, continuing into ( b ) the left and right bundle braches, which show intense labeling of Na v 1.5 and desmin, but absence of Cx43 ( LBB left bundle branch, RBB right bundle branch, rv right ventricle, ivs intraventricular septum). c Serial sections of Purkinje fibers ( PF ) show high Na v 1.5, HCN4, and Cx40 labeling intensity, but no Cx43. d In situ hybridization of adult heart shows similar high expression levels of Hcn4 and Scn5a mRNA in AVB ( blue signals ) in comparison with the myocardium of the intraventricular septum ( ivs )

Article Snippet: Antibodies The following primary antibodies were used: rabbit polyclonal anti-Nav1.5 (1:200, Alomone Laboratories, ASC-005), mouse monoclonal anti-alpha-actinin (1:1000, Sigma), mouse monoclonal anti-desmin (1:200, Monosan, MON-3001), mouse monoclonal anti-Cx43 (1:200, BD Biosciences, 610061), rabbit polyclonal anti-Cx40 (1:250, Chemicon), rabbit polyclonal anti-HCN4 (1:200, Chemicon, AB5808), and rabbit polyclonal anti-cardiac troponin I (anti-cTnI, 1:1000, HytestLtd).

Techniques: Expressing, Labeling, In Situ Hybridization

Journal: Basic Research in Cardiology

Article Title: The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

doi: 10.1007/s00395-009-0012-8

Figure Lengend Snippet: Transmural distribution of Na v 1.5 in the ventricular myocardium. Low Na v 1.5 labeling intensity in ventricular epicardium ( arrowheads ) compared to the midmyocardial layer ( m ) in ( a ) the apex and ( b ) left ventricular free wall of the adult mouse heart. For comparison, α-actinin and desmin show a homogeneous transmural labeling. c Left panel shows low Na v 1.5 expression in subepicardium ( arrowheads ) and high Na v 1.5 expression in subendocardium ( arrows ) of the right ventricle. For comparison, Cx43 shows a homogeneous transmural distribution. Also shown are details of subendocardial surface ( middle panel ) and subepicardial region ( right panel ) of the right ventricle. d In situ hybridization of adult ventricular myocardium shows clear Scn5a staining ( in blue ) in the subendocardium ( arrows ) and low Scn5a expression in the subepicardium ( asterisks ). The right panel depicts a magnified section of the left panel indicating higher Scn5a expression in the subendocardium ( arrows ) compared to midmural ( m )

Article Snippet: Antibodies The following primary antibodies were used: rabbit polyclonal anti-Nav1.5 (1:200, Alomone Laboratories, ASC-005), mouse monoclonal anti-alpha-actinin (1:1000, Sigma), mouse monoclonal anti-desmin (1:200, Monosan, MON-3001), mouse monoclonal anti-Cx43 (1:200, BD Biosciences, 610061), rabbit polyclonal anti-Cx40 (1:250, Chemicon), rabbit polyclonal anti-HCN4 (1:200, Chemicon, AB5808), and rabbit polyclonal anti-cardiac troponin I (anti-cTnI, 1:1000, HytestLtd).

Techniques: Labeling, Expressing, In Situ Hybridization, Staining

Journal: Basic Research in Cardiology

Article Title: The cardiac sodium channel displays differential distribution in the conduction system and transmural heterogeneity in the murine ventricular myocardium

doi: 10.1007/s00395-009-0012-8

Figure Lengend Snippet: Distribution of Na v 1.5 and Scn5a in the conduction system in embryonic hearts (ED14.5). a High expression levels of Na v 1.5 protein in the AV bundle ( AVB ) and bundle branches ( BB ) in comparison with the ventricular myocardium of the intraventricular septum ( ivs ); the right panel represents an enlarged section of the left panel . b In situ hybridization shows prominent Scn5a mRNA expression ( in blue ) but absence of Cx43 mRNA in the AVB ( dashed line indicates region of AVB)

Article Snippet: Antibodies The following primary antibodies were used: rabbit polyclonal anti-Nav1.5 (1:200, Alomone Laboratories, ASC-005), mouse monoclonal anti-alpha-actinin (1:1000, Sigma), mouse monoclonal anti-desmin (1:200, Monosan, MON-3001), mouse monoclonal anti-Cx43 (1:200, BD Biosciences, 610061), rabbit polyclonal anti-Cx40 (1:250, Chemicon), rabbit polyclonal anti-HCN4 (1:200, Chemicon, AB5808), and rabbit polyclonal anti-cardiac troponin I (anti-cTnI, 1:1000, HytestLtd).

Techniques: Expressing, In Situ Hybridization