hcn4  (Alomone Labs)


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    Alomone Labs hcn4
    Transcriptional profiling of Cx30.2-lacZ + cells identifies MyoR as an AVC-enriched transcript. (A) Strategy used for purifying E12.5 Cx30.2-lacZ + AVC cells by flow cytometry. (B) Partial list of genes found to be enriched in microarray analysis. Fold enrichment refers to transcript levels in Cx30.2-lacZ + cells relative to lacZ -null cells. Note that MyoR transcripts were 7-fold more abundant in Cx30.2-lacZ + cells within the E12.5 heart. (C) qRT-PCR analysis of microdissected E10.5 AVC tissue demonstrating enrichment of MyoR. Fold enrichment refers to transcript levels in dissected AVC tissue versus whole heart. (D) Developmental analysis of MyoR expression by qRT-PCR analysis. MyoR expression in the whole heart at a particular time point relative to E10.5 is shown. MyoR transcripts were found to peak during late gestation (E16.5). (E) qRT-PCR analysis of MyoR expression level in Cx30.2-EGFP + AVC cells isolated at E16.5. MyoR expression is shown relative to transcript levels in EGFP − AVC cells. (F) Western blot analysis of AVC and ventricular nuclear extracts dissected from E16.5 embryos (top) with the indicated antibodies. Band quantitation and normalization demonstrated ∼14-fold enrichment of MyoR in the E16.5 AVC relative to ventricular tissue. (G) Immunostaining of consecutive sections through an E16.5 Cx30.2-EGFP heart for GFP, Gata4, <t>Hcn4,</t> and MyoR, demonstrating localization of Cx30.2, Gata4, and MyoR to the Hcn4 + AVN (dashed outline). All sections were counterstained with DAPI to stain cell nuclei. Scale bar, 20 μm. LV, left ventricle; RV, right ventricle; FDG, fluorescein digalactopyranoside; AVC, atrioventricular canal; AVN, atrioventricular node.
    Hcn4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    hcn4 - by Bioz Stars, 2022-10
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    Images

    1) Product Images from "MyoR Modulates Cardiac Conduction by Repressing Gata4"

    Article Title: MyoR Modulates Cardiac Conduction by Repressing Gata4

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00860-14

    Transcriptional profiling of Cx30.2-lacZ + cells identifies MyoR as an AVC-enriched transcript. (A) Strategy used for purifying E12.5 Cx30.2-lacZ + AVC cells by flow cytometry. (B) Partial list of genes found to be enriched in microarray analysis. Fold enrichment refers to transcript levels in Cx30.2-lacZ + cells relative to lacZ -null cells. Note that MyoR transcripts were 7-fold more abundant in Cx30.2-lacZ + cells within the E12.5 heart. (C) qRT-PCR analysis of microdissected E10.5 AVC tissue demonstrating enrichment of MyoR. Fold enrichment refers to transcript levels in dissected AVC tissue versus whole heart. (D) Developmental analysis of MyoR expression by qRT-PCR analysis. MyoR expression in the whole heart at a particular time point relative to E10.5 is shown. MyoR transcripts were found to peak during late gestation (E16.5). (E) qRT-PCR analysis of MyoR expression level in Cx30.2-EGFP + AVC cells isolated at E16.5. MyoR expression is shown relative to transcript levels in EGFP − AVC cells. (F) Western blot analysis of AVC and ventricular nuclear extracts dissected from E16.5 embryos (top) with the indicated antibodies. Band quantitation and normalization demonstrated ∼14-fold enrichment of MyoR in the E16.5 AVC relative to ventricular tissue. (G) Immunostaining of consecutive sections through an E16.5 Cx30.2-EGFP heart for GFP, Gata4, Hcn4, and MyoR, demonstrating localization of Cx30.2, Gata4, and MyoR to the Hcn4 + AVN (dashed outline). All sections were counterstained with DAPI to stain cell nuclei. Scale bar, 20 μm. LV, left ventricle; RV, right ventricle; FDG, fluorescein digalactopyranoside; AVC, atrioventricular canal; AVN, atrioventricular node.
    Figure Legend Snippet: Transcriptional profiling of Cx30.2-lacZ + cells identifies MyoR as an AVC-enriched transcript. (A) Strategy used for purifying E12.5 Cx30.2-lacZ + AVC cells by flow cytometry. (B) Partial list of genes found to be enriched in microarray analysis. Fold enrichment refers to transcript levels in Cx30.2-lacZ + cells relative to lacZ -null cells. Note that MyoR transcripts were 7-fold more abundant in Cx30.2-lacZ + cells within the E12.5 heart. (C) qRT-PCR analysis of microdissected E10.5 AVC tissue demonstrating enrichment of MyoR. Fold enrichment refers to transcript levels in dissected AVC tissue versus whole heart. (D) Developmental analysis of MyoR expression by qRT-PCR analysis. MyoR expression in the whole heart at a particular time point relative to E10.5 is shown. MyoR transcripts were found to peak during late gestation (E16.5). (E) qRT-PCR analysis of MyoR expression level in Cx30.2-EGFP + AVC cells isolated at E16.5. MyoR expression is shown relative to transcript levels in EGFP − AVC cells. (F) Western blot analysis of AVC and ventricular nuclear extracts dissected from E16.5 embryos (top) with the indicated antibodies. Band quantitation and normalization demonstrated ∼14-fold enrichment of MyoR in the E16.5 AVC relative to ventricular tissue. (G) Immunostaining of consecutive sections through an E16.5 Cx30.2-EGFP heart for GFP, Gata4, Hcn4, and MyoR, demonstrating localization of Cx30.2, Gata4, and MyoR to the Hcn4 + AVN (dashed outline). All sections were counterstained with DAPI to stain cell nuclei. Scale bar, 20 μm. LV, left ventricle; RV, right ventricle; FDG, fluorescein digalactopyranoside; AVC, atrioventricular canal; AVN, atrioventricular node.

    Techniques Used: Flow Cytometry, Cytometry, Microarray, Quantitative RT-PCR, Expressing, Isolation, Western Blot, Quantitation Assay, Immunostaining, Staining

    2) Product Images from "Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue"

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    Journal: bioRxiv

    doi: 10.1101/2020.04.14.039461

    Panels A and B : Optical slices (1 μm thick) of HCN4 immunoreactive cells at a depth of 30 μm and 60 μm from the endocardial surface. Panel C : a 3D reconstruction of 70 stacked HCN4 images (optically sliced via confocal microscope) of an immunolabeled whole-mount SAN preparation demonstrating the distribution of HCN4 immunoreactive cells within a depth of 70 μM from the endocardium. Tissue depth from the endocardial site is color coded on the right side of the panel.
    Figure Legend Snippet: Panels A and B : Optical slices (1 μm thick) of HCN4 immunoreactive cells at a depth of 30 μm and 60 μm from the endocardial surface. Panel C : a 3D reconstruction of 70 stacked HCN4 images (optically sliced via confocal microscope) of an immunolabeled whole-mount SAN preparation demonstrating the distribution of HCN4 immunoreactive cells within a depth of 70 μM from the endocardium. Tissue depth from the endocardial site is color coded on the right side of the panel.

    Techniques Used: Microscopy, Immunolabeling

    A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Stacked confocal images and reconstructed front view of optically sliced z-stack images of CX43 at a depth of 35 μm from endocardium. Gap junctions are color coded by depth and plotted within the z-stacks reconstructed from optical slices. Confocal images were acquired with a 40x oil immersion. Panel B: Stacked confocal images and reconstructed side view of optically sliced z-stack images of HCN4 + /F-actin - /(CX43) - cells (red) and HCN4 - /F-actin + /(CX43) + cells (cyan) at a depth of 40 μm and 50 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective Panel C: Optical slice shows HCN4 + /F-actin - /(CX43) - cells (red) adjacent to HCN4 - /F-actin + /(CX43) + cells (cyan). CX43 protein (green) is expressed only in cyan cells.
    Figure Legend Snippet: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Stacked confocal images and reconstructed front view of optically sliced z-stack images of CX43 at a depth of 35 μm from endocardium. Gap junctions are color coded by depth and plotted within the z-stacks reconstructed from optical slices. Confocal images were acquired with a 40x oil immersion. Panel B: Stacked confocal images and reconstructed side view of optically sliced z-stack images of HCN4 + /F-actin - /(CX43) - cells (red) and HCN4 - /F-actin + /(CX43) + cells (cyan) at a depth of 40 μm and 50 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective Panel C: Optical slice shows HCN4 + /F-actin - /(CX43) - cells (red) adjacent to HCN4 - /F-actin + /(CX43) + cells (cyan). CX43 protein (green) is expressed only in cyan cells.

    Techniques Used: Immunolabeling

    Panel A and B: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 36 tiled confocal images of the area of the 1350μm by 1350μm demonstrate meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. Gray color tissue was imaged in transmitted light. Panel B: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 16 tiled confocal images of the area of the 900μm by 900μm of another area of meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. CX43 is detected only in F-actin labeled cells.
    Figure Legend Snippet: Panel A and B: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 36 tiled confocal images of the area of the 1350μm by 1350μm demonstrate meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. Gray color tissue was imaged in transmitted light. Panel B: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 16 tiled confocal images of the area of the 900μm by 900μm of another area of meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. CX43 is detected only in F-actin labeled cells.

    Techniques Used: Immunolabeling, Labeling

    Panel A : An immunolabelled, whole mount image of a SAN preparation at low (2.5x) optical magnification. Panel B: Stacked confocal images and reconstructed front view of optically sliced z-stack images of HCN4 + /F-actin - cells (red) and HCN4 - /F-actin + cells (cyan) at a depth of 30 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective within ROI (yellow) shown in panel A. Panel C: Side views of the z-stack images in panel B, illustrating the intertwining cells of HCN4-meshwork and F-actin networks across the 30 μm depth.
    Figure Legend Snippet: Panel A : An immunolabelled, whole mount image of a SAN preparation at low (2.5x) optical magnification. Panel B: Stacked confocal images and reconstructed front view of optically sliced z-stack images of HCN4 + /F-actin - cells (red) and HCN4 - /F-actin + cells (cyan) at a depth of 30 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective within ROI (yellow) shown in panel A. Panel C: Side views of the z-stack images in panel B, illustrating the intertwining cells of HCN4-meshwork and F-actin networks across the 30 μm depth.

    Techniques Used:

    Panel A: An Image of a whole mount SAN preparation at low (2.5x) optical magnification, demonstrating the distribution of HCN4 (red color) immunoreactive and F-actin (cyan color) labelled cells. The merged images between HCN4 and F-actin is shown in both panels. Panel B: Tiled Image of the HCN4 + /F-actin - cell meshwork (red) intertwined with the HCN4 - /F-actin + cell network (cyan) reconstructed from 4 images recorded via 10x water immersion objective within the red box in panel A.
    Figure Legend Snippet: Panel A: An Image of a whole mount SAN preparation at low (2.5x) optical magnification, demonstrating the distribution of HCN4 (red color) immunoreactive and F-actin (cyan color) labelled cells. The merged images between HCN4 and F-actin is shown in both panels. Panel B: Tiled Image of the HCN4 + /F-actin - cell meshwork (red) intertwined with the HCN4 - /F-actin + cell network (cyan) reconstructed from 4 images recorded via 10x water immersion objective within the red box in panel A.

    Techniques Used:

    Upper panels - HCN4 immunoreactive SAN cells: elongated (magenta arrows in Panel A ), novel, pyramidal-like shape cells (yellow arrow in Panels B and D ), spider-like ( Panel C ), and spindle cells ( Panel E , blue arrow). Lower panels - SAN cells loaded with Fluo-4 AM have similar shapes to immunolabelled HCN4 + cells in the upper panel. Spider-like cells ( Panel F ) are indicated by the red arrow. Novel cells with a pyramidal-like soma ( Panel G ) are indicated by yellow arrows. Spindle cells are indicated by the blue arrow ( Panel G and H ). Elongated cells ( Panel H ) are indicated by magenta arrows.
    Figure Legend Snippet: Upper panels - HCN4 immunoreactive SAN cells: elongated (magenta arrows in Panel A ), novel, pyramidal-like shape cells (yellow arrow in Panels B and D ), spider-like ( Panel C ), and spindle cells ( Panel E , blue arrow). Lower panels - SAN cells loaded with Fluo-4 AM have similar shapes to immunolabelled HCN4 + cells in the upper panel. Spider-like cells ( Panel F ) are indicated by the red arrow. Novel cells with a pyramidal-like soma ( Panel G ) are indicated by yellow arrows. Spindle cells are indicated by the blue arrow ( Panel G and H ). Elongated cells ( Panel H ) are indicated by magenta arrows.

    Techniques Used:

    Panel A: A dual immunolabeled HCN4 (red) and CX43 (green) whole mount SAN image at low optical magnification (2.5x). Merged (CX43 and HCN4) immunoreactivity is shown in all three panels. Panel B: Image within the ROI in panel A reconstructed from 4 tile images of the HCN4 + /CX43 - meshwork (red) intertwined with HCN4 - /(CX43) + network (green) taken with 10x water immersion objective. Panels C : Confocal images from the area within the ROI in panel B showing: HCN4 + cells that do not express CX43 (upper image); intertwining areas between HCN4 + /(Cx43) - meshwork (red color), and penetrating HCN4-/ CX43 + cells outlined by green dots corresponding to CX43 protein on the cell membranes (middle and lower panel). Note that HCN4 expressing cells in all three images do not express CX43.
    Figure Legend Snippet: Panel A: A dual immunolabeled HCN4 (red) and CX43 (green) whole mount SAN image at low optical magnification (2.5x). Merged (CX43 and HCN4) immunoreactivity is shown in all three panels. Panel B: Image within the ROI in panel A reconstructed from 4 tile images of the HCN4 + /CX43 - meshwork (red) intertwined with HCN4 - /(CX43) + network (green) taken with 10x water immersion objective. Panels C : Confocal images from the area within the ROI in panel B showing: HCN4 + cells that do not express CX43 (upper image); intertwining areas between HCN4 + /(Cx43) - meshwork (red color), and penetrating HCN4-/ CX43 + cells outlined by green dots corresponding to CX43 protein on the cell membranes (middle and lower panel). Note that HCN4 expressing cells in all three images do not express CX43.

    Techniques Used: Immunolabeling, Expressing

    3) Product Images from "Proteolytic Processing of HCN2 and Co-assembly with HCN4 in the Generation of Cardiac Pacemaker Channels *"

    Article Title: Proteolytic Processing of HCN2 and Co-assembly with HCN4 in the Generation of Cardiac Pacemaker Channels *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.007583

    A C-terminal-truncated HCN2 (HCN2ΔC) protein forms heteromeric channels with HCN4 in HEK-293 cells. A , representative recordings obtained from (wild type or HCN4) expressing HEK-293 cells following transient transfection with cDNA constructs encoding the full-length HCN2 or the C-terminal HCN2 truncation in mutant, HCN2ΔC. Representative currents in HEK-293 cells stably expressing HCN4 are also illustrated. In each case, currents evoked during 3 s hyperpolarizing voltage steps to test potentials between −130 and −60 mV from a holding potential of −40 mV are illustrated; the voltage clamp paradigm is illustrated below the records. B , peak currents at each test potential in each cell were measured and normalized to the maximal peak current recorded in the same cell, and mean ± S.E. normalized peak currents are plotted as a function of the test potential; the individual curves were fitted to the Boltzmann equation (see “Materials and Methods”). Mean ± S.E. voltages (V 1/2 ) of half-maximal current activation ( C ) and activation time constants ( D ) are illustrated; n values are presented above the bars (*, p
    Figure Legend Snippet: A C-terminal-truncated HCN2 (HCN2ΔC) protein forms heteromeric channels with HCN4 in HEK-293 cells. A , representative recordings obtained from (wild type or HCN4) expressing HEK-293 cells following transient transfection with cDNA constructs encoding the full-length HCN2 or the C-terminal HCN2 truncation in mutant, HCN2ΔC. Representative currents in HEK-293 cells stably expressing HCN4 are also illustrated. In each case, currents evoked during 3 s hyperpolarizing voltage steps to test potentials between −130 and −60 mV from a holding potential of −40 mV are illustrated; the voltage clamp paradigm is illustrated below the records. B , peak currents at each test potential in each cell were measured and normalized to the maximal peak current recorded in the same cell, and mean ± S.E. normalized peak currents are plotted as a function of the test potential; the individual curves were fitted to the Boltzmann equation (see “Materials and Methods”). Mean ± S.E. voltages (V 1/2 ) of half-maximal current activation ( C ) and activation time constants ( D ) are illustrated; n values are presented above the bars (*, p

    Techniques Used: Expressing, Transfection, Construct, Mutagenesis, Stable Transfection, Activation Assay

    Co-immunoprecipitation of HCN2 and HCN4 from adult (mouse) heart. Proteins were isolated from adult mouse heart and immunoprecipitations ( IPs ) were performed with the rabbit IgG ( control ), anti-N-HCN4 ( A ), or anti-N2-HCN2 ( B ) polyclonal antibody. Following the immunoprecipitations, proteins were eluted from the beads, fractionated on polyacrylamide (SDS-PAGE) gels, and Western blots ( IB ) were performed using either the anti-N2-HCN2 ( A ) or the anti-N-HCN4 ( B ) polyclonal antibody. Either HCN2 protein bands or HCN4 protein bands were indicated by a closed arrow . IgG bands were indicated by an open arrow . Although full-length (160 kDa) HCN4 protein was readily identified in the heart lysate, i.e. prior to the immunoprecipitation ( lane 4 ) and in the (immunoprecipitated) protein sample obtained following the immunoprecipitation with the anti-N2-HCN2 antibody ( B ), the anti-N2-HCN2 antibody detected only a single low molecular mass ∼60-kDA protein ( A ) in both the immunoprecipitated protein sample and in the Western blot of the whole heart lysate. No additional HCN2 ( A ) or HCN4 ( B ) proteins were observed in the samples obtained following further washing of the beads ( lanes A3 and B3 ). As is also evident, neither HCN2 ( A ) nor HCN4 ( B ) was immunoprecipitated using rabbit IgG ( lanes A1 and B1 ).
    Figure Legend Snippet: Co-immunoprecipitation of HCN2 and HCN4 from adult (mouse) heart. Proteins were isolated from adult mouse heart and immunoprecipitations ( IPs ) were performed with the rabbit IgG ( control ), anti-N-HCN4 ( A ), or anti-N2-HCN2 ( B ) polyclonal antibody. Following the immunoprecipitations, proteins were eluted from the beads, fractionated on polyacrylamide (SDS-PAGE) gels, and Western blots ( IB ) were performed using either the anti-N2-HCN2 ( A ) or the anti-N-HCN4 ( B ) polyclonal antibody. Either HCN2 protein bands or HCN4 protein bands were indicated by a closed arrow . IgG bands were indicated by an open arrow . Although full-length (160 kDa) HCN4 protein was readily identified in the heart lysate, i.e. prior to the immunoprecipitation ( lane 4 ) and in the (immunoprecipitated) protein sample obtained following the immunoprecipitation with the anti-N2-HCN2 antibody ( B ), the anti-N2-HCN2 antibody detected only a single low molecular mass ∼60-kDA protein ( A ) in both the immunoprecipitated protein sample and in the Western blot of the whole heart lysate. No additional HCN2 ( A ) or HCN4 ( B ) proteins were observed in the samples obtained following further washing of the beads ( lanes A3 and B3 ). As is also evident, neither HCN2 ( A ) nor HCN4 ( B ) was immunoprecipitated using rabbit IgG ( lanes A1 and B1 ).

    Techniques Used: Immunoprecipitation, Isolation, SDS Page, Western Blot

    Detection of full-length channel proteins in heart lysates. Proteins, isolated from HEK-293 cells stably expressing HCN4 and from adult mouse heart, were fractionated on SDS-PAGE gels and immunoblotted ( IB ) with the anti-N-HCN4 antibody ( A ) or the anti-C-HCN4 antibody ( B ). HCN4 and Nav1.5 protein bands were indicated by a closed arrow . A ∼160-kDa band was detected in samples from the HEK-293 cell line stably expressing HCN4 and in mouse heart. Nothing was detected in untransfected (wild type) HEK-293 cells with either of these (anti-HCN4) antibodies. C , similarly, a single, high molecular mass protein (at ∼250 kDa) was detected in lysates from adult mouse heart and from HEK-293 cells stably expressing Nav1.5, but not in extracts from untransfected HEK-293 cells.
    Figure Legend Snippet: Detection of full-length channel proteins in heart lysates. Proteins, isolated from HEK-293 cells stably expressing HCN4 and from adult mouse heart, were fractionated on SDS-PAGE gels and immunoblotted ( IB ) with the anti-N-HCN4 antibody ( A ) or the anti-C-HCN4 antibody ( B ). HCN4 and Nav1.5 protein bands were indicated by a closed arrow . A ∼160-kDa band was detected in samples from the HEK-293 cell line stably expressing HCN4 and in mouse heart. Nothing was detected in untransfected (wild type) HEK-293 cells with either of these (anti-HCN4) antibodies. C , similarly, a single, high molecular mass protein (at ∼250 kDa) was detected in lysates from adult mouse heart and from HEK-293 cells stably expressing Nav1.5, but not in extracts from untransfected HEK-293 cells.

    Techniques Used: Isolation, Stable Transfection, Expressing, SDS Page

    4) Product Images from "Spatiotemporal Regulation of an Hcn4 Enhancer Defines a Role for Mef2c and HDACs in Cardiac Electrical Patterning"

    Article Title: Spatiotemporal Regulation of an Hcn4 Enhancer Defines a Role for Mef2c and HDACs in Cardiac Electrical Patterning

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2012.10.017

    Identification of minimal enhancer regions within R2R3 that direct reporter activity in the Hcn4 expression domain
    Figure Legend Snippet: Identification of minimal enhancer regions within R2R3 that direct reporter activity in the Hcn4 expression domain

    Techniques Used: Activity Assay, Expressing

    HDAC Activity Regulates Hcn4 R2R3
    Figure Legend Snippet: HDAC Activity Regulates Hcn4 R2R3

    Techniques Used: Activity Assay

    Mef2C regulates R2R3-LacZ and Hcn4 expression in vivo
    Figure Legend Snippet: Mef2C regulates R2R3-LacZ and Hcn4 expression in vivo

    Techniques Used: Expressing, In Vivo

    Identification and in vivo testing of Hcn4 regulatory elements
    Figure Legend Snippet: Identification and in vivo testing of Hcn4 regulatory elements

    Techniques Used: In Vivo

    In vivo temporal reporter activity directed by the combined R2-R3 Hcn4 Enhancers
    Figure Legend Snippet: In vivo temporal reporter activity directed by the combined R2-R3 Hcn4 Enhancers

    Techniques Used: In Vivo, Activity Assay

    In vivo expression directed by the R2R3 Hcn4 enhancer in the post-natal AV bundle and its interface with the AV Node
    Figure Legend Snippet: In vivo expression directed by the R2R3 Hcn4 enhancer in the post-natal AV bundle and its interface with the AV Node

    Techniques Used: In Vivo, Expressing

    Mef2C regulates R2R3 Hcn4 enhancer activity via a conserved Mef2 Site
    Figure Legend Snippet: Mef2C regulates R2R3 Hcn4 enhancer activity via a conserved Mef2 Site

    Techniques Used: Activity Assay

    Expression pattern of Hcn4 mRNA by whole-mount in-situ hybridization
    Figure Legend Snippet: Expression pattern of Hcn4 mRNA by whole-mount in-situ hybridization

    Techniques Used: Expressing, In Situ Hybridization

    5) Product Images from "Programming and Isolation of Highly Pure Physiologically and Pharmacologically Functional Sinus-Nodal Bodies from Pluripotent Stem Cells"

    Article Title: Programming and Isolation of Highly Pure Physiologically and Pharmacologically Functional Sinus-Nodal Bodies from Pluripotent Stem Cells

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2014.03.006

    Electrophysiological Parameters of Pacemaker-like Cardiomyocytes Obtained from iSABs (A) Distribution of pacemaker cells as shown by single-cell patch-clamp and funny-channel measurements: > 81% of pacemaker-like cells were achieved; 19% of these represented immature pacemaker cells and the rest were mature pacemaker cells (n = 65). The data represent seven independent experiments and are presented as means ± SD. See also Tables S1–S3 . (B) Patch-clamp protocol. Voltage was applied to elicit the hyperpolarization-activated current for I f recordings from isolated iSAB cells. (C) Example of I f current, recorded from an isolated iSAB-derived cell. (D and E) Current density (D) and time constant of activation (E) at –130 mV, demonstrating a robust I f expression with slow activation kinetics typical of the HCN4 channel subtype and mature SA nodal I f (n = 17). The data represent three independent experiments and are presented as means ± SD. (F) Reaction to β-adrenergic (isoprotenerol) and muscarinic (carbachol) stimulation leads to typical accelerated versus decelerated AP rates. iSAB-derived cells show a pronounced response to isoprotenerol, with beating rates reaching up to 560 bpm. iSAB/Iso: n = 9; iSAB/Carb: n = 5. The data represent seven independent experiments and are presented as means ± SD. See also Figures S1 and S2 .
    Figure Legend Snippet: Electrophysiological Parameters of Pacemaker-like Cardiomyocytes Obtained from iSABs (A) Distribution of pacemaker cells as shown by single-cell patch-clamp and funny-channel measurements: > 81% of pacemaker-like cells were achieved; 19% of these represented immature pacemaker cells and the rest were mature pacemaker cells (n = 65). The data represent seven independent experiments and are presented as means ± SD. See also Tables S1–S3 . (B) Patch-clamp protocol. Voltage was applied to elicit the hyperpolarization-activated current for I f recordings from isolated iSAB cells. (C) Example of I f current, recorded from an isolated iSAB-derived cell. (D and E) Current density (D) and time constant of activation (E) at –130 mV, demonstrating a robust I f expression with slow activation kinetics typical of the HCN4 channel subtype and mature SA nodal I f (n = 17). The data represent three independent experiments and are presented as means ± SD. (F) Reaction to β-adrenergic (isoprotenerol) and muscarinic (carbachol) stimulation leads to typical accelerated versus decelerated AP rates. iSAB-derived cells show a pronounced response to isoprotenerol, with beating rates reaching up to 560 bpm. iSAB/Iso: n = 9; iSAB/Carb: n = 5. The data represent seven independent experiments and are presented as means ± SD. See also Figures S1 and S2 .

    Techniques Used: Patch Clamp, Isolation, Derivative Assay, Activation Assay, Expressing

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    Alomone Labs rabbit anti hcn4
    The tdTomato-expressing retinal bipolar cells in the 5-HTR2a-cre mouse line are co-labeled with antibodies specific to type 4 and type 3b cone bipolar cells, and rod bipolar cells. A – C : In a retinal vertical section, the tdTomato-expressing retina ( A ) was immunostained for calsenilin ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells are marked with stars. D – F : In a retinal whole mount with the focal plane at the distal portion of the inner nuclear layer (INL), the tdTomato-expressing retina ( D ) was immunostained for calsenilin ( E ). The overlay of D and E is shown in F . The majority of the tomato-expressing cells in the distal portion of the INL are calsenilin-positive. The tdTomato-expressing cells that do not show calsenilin staining are marked with arrowheads. The tdTomato-expressing retina was immunostained for PKCα in a retinal vertical section ( G–I ). The double-positive bipolar cells are marked with arrows in the somata and arrowheads pointing to the axon terminals. J – L : The tdTomato-expressing retina was immunostained for PKARIIβ. Two double-positive cells are marked with stars. The tdTomato-expressing retinal bipolar cells were not labeled by antibodies for <t>HCN4</t> ( M – O ) and Syt2 ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
    Rabbit Anti Hcn4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    rabbit anti hcn4 - by Bioz Stars, 2022-10
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    The tdTomato-expressing retinal bipolar cells in the 5-HTR2a-cre mouse line are co-labeled with antibodies specific to type 4 and type 3b cone bipolar cells, and rod bipolar cells. A – C : In a retinal vertical section, the tdTomato-expressing retina ( A ) was immunostained for calsenilin ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells are marked with stars. D – F : In a retinal whole mount with the focal plane at the distal portion of the inner nuclear layer (INL), the tdTomato-expressing retina ( D ) was immunostained for calsenilin ( E ). The overlay of D and E is shown in F . The majority of the tomato-expressing cells in the distal portion of the INL are calsenilin-positive. The tdTomato-expressing cells that do not show calsenilin staining are marked with arrowheads. The tdTomato-expressing retina was immunostained for PKCα in a retinal vertical section ( G–I ). The double-positive bipolar cells are marked with arrows in the somata and arrowheads pointing to the axon terminals. J – L : The tdTomato-expressing retina was immunostained for PKARIIβ. Two double-positive cells are marked with stars. The tdTomato-expressing retinal bipolar cells were not labeled by antibodies for HCN4 ( M – O ) and Syt2 ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Journal: Molecular Vision

    Article Title: Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre transgenic mouse lines

    doi:

    Figure Lengend Snippet: The tdTomato-expressing retinal bipolar cells in the 5-HTR2a-cre mouse line are co-labeled with antibodies specific to type 4 and type 3b cone bipolar cells, and rod bipolar cells. A – C : In a retinal vertical section, the tdTomato-expressing retina ( A ) was immunostained for calsenilin ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells are marked with stars. D – F : In a retinal whole mount with the focal plane at the distal portion of the inner nuclear layer (INL), the tdTomato-expressing retina ( D ) was immunostained for calsenilin ( E ). The overlay of D and E is shown in F . The majority of the tomato-expressing cells in the distal portion of the INL are calsenilin-positive. The tdTomato-expressing cells that do not show calsenilin staining are marked with arrowheads. The tdTomato-expressing retina was immunostained for PKCα in a retinal vertical section ( G–I ). The double-positive bipolar cells are marked with arrows in the somata and arrowheads pointing to the axon terminals. J – L : The tdTomato-expressing retina was immunostained for PKARIIβ. Two double-positive cells are marked with stars. The tdTomato-expressing retinal bipolar cells were not labeled by antibodies for HCN4 ( M – O ) and Syt2 ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Article Snippet: The following antibodies were used in this study: rabbit anti-mCherry (1:500; 632,496, Clontech, Mountain View, CA); mouse anticalsenilin (1:2,000; kindly provided by W. Wasco, Harvard Medical School, Boston, MA); rabbit anti-PKC (1:20,000; catalog number 2056, Cell Signal, Danvers, MA); mouse anti-PKC (1:10000; catalog number sc8393, Santa Cruz, CA); mouse antisynaptotagmin II (Syt2; 1:600; Zebrafish International Resource Center, Eugene, OR); rabbit anti-HCN4 (1:500; Alomone Labs, Jerusalem, Israel); and mouse antiprotein kinase A (PKA) RIIβ (1:80,000; BD Biosciences, San Jose, CA).

    Techniques: Expressing, Labeling, Staining

    The tdTomato-expressing retinal bipolar cells in the Pcp2-cre mouse line are co-labeled with antibodies specific to rod bipolar cells, type 2 and 6 cone bipolar cells. A – C : In retinal vertical sections, the tdTomato-expressing retina ( A ) was immunostained for PKCα ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells were marked with stars in the somata and with arrowheads pointing at the axon terminals. D–F : In the retinal whole mount with the focal plane in the INL, colabeling with tdTomato and PKCα. PKCα-negative tdTomato-expressing cells are marked with arrows. G–I : The tdTomato-expressing retina was immunostained for Syt2. The double-positive bipolar cells with axon terminals stratified at the distal portion of the IPL (type 2 bipolar cells) are marked with white stars in the somata and with white arrowheads pointing at the axon terminals. The double-positive bipolar cells with axon terminals stratified in the proximal portion of the IPL (type 6 bipolar cells) are marked with yellow arrows at the somata and yellow arrowheads at the axon terminals. A tdTomato-expressing bipolar cell with their axon terminals stratified slightly distal to Syt2-positive cells is marked with a blue arrowhead ( I ). The tdTomato-expressing retinal bipolar cells were not found to be labeled by PKARIIβ ( J – L ), HCN4 ( M – O ), and calsenilin ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Journal: Molecular Vision

    Article Title: Cre-mediated recombination efficiency and transgene expression patterns of three retinal bipolar cell-expressing Cre transgenic mouse lines

    doi:

    Figure Lengend Snippet: The tdTomato-expressing retinal bipolar cells in the Pcp2-cre mouse line are co-labeled with antibodies specific to rod bipolar cells, type 2 and 6 cone bipolar cells. A – C : In retinal vertical sections, the tdTomato-expressing retina ( A ) was immunostained for PKCα ( B ). The overlay of A and B is shown in C . The double-positive bipolar cells were marked with stars in the somata and with arrowheads pointing at the axon terminals. D–F : In the retinal whole mount with the focal plane in the INL, colabeling with tdTomato and PKCα. PKCα-negative tdTomato-expressing cells are marked with arrows. G–I : The tdTomato-expressing retina was immunostained for Syt2. The double-positive bipolar cells with axon terminals stratified at the distal portion of the IPL (type 2 bipolar cells) are marked with white stars in the somata and with white arrowheads pointing at the axon terminals. The double-positive bipolar cells with axon terminals stratified in the proximal portion of the IPL (type 6 bipolar cells) are marked with yellow arrows at the somata and yellow arrowheads at the axon terminals. A tdTomato-expressing bipolar cell with their axon terminals stratified slightly distal to Syt2-positive cells is marked with a blue arrowhead ( I ). The tdTomato-expressing retinal bipolar cells were not found to be labeled by PKARIIβ ( J – L ), HCN4 ( M – O ), and calsenilin ( P – R ). Scale bars represent 50 µm. ONL, outer nuclear layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.

    Article Snippet: The following antibodies were used in this study: rabbit anti-mCherry (1:500; 632,496, Clontech, Mountain View, CA); mouse anticalsenilin (1:2,000; kindly provided by W. Wasco, Harvard Medical School, Boston, MA); rabbit anti-PKC (1:20,000; catalog number 2056, Cell Signal, Danvers, MA); mouse anti-PKC (1:10000; catalog number sc8393, Santa Cruz, CA); mouse antisynaptotagmin II (Syt2; 1:600; Zebrafish International Resource Center, Eugene, OR); rabbit anti-HCN4 (1:500; Alomone Labs, Jerusalem, Israel); and mouse antiprotein kinase A (PKA) RIIβ (1:80,000; BD Biosciences, San Jose, CA).

    Techniques: Expressing, Labeling

    Panels A and B : Optical slices (1 μm thick) of HCN4 immunoreactive cells at a depth of 30 μm and 60 μm from the endocardial surface. Panel C : a 3D reconstruction of 70 stacked HCN4 images (optically sliced via confocal microscope) of an immunolabeled whole-mount SAN preparation demonstrating the distribution of HCN4 immunoreactive cells within a depth of 70 μM from the endocardium. Tissue depth from the endocardial site is color coded on the right side of the panel.

    Journal: bioRxiv

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    doi: 10.1101/2020.04.14.039461

    Figure Lengend Snippet: Panels A and B : Optical slices (1 μm thick) of HCN4 immunoreactive cells at a depth of 30 μm and 60 μm from the endocardial surface. Panel C : a 3D reconstruction of 70 stacked HCN4 images (optically sliced via confocal microscope) of an immunolabeled whole-mount SAN preparation demonstrating the distribution of HCN4 immunoreactive cells within a depth of 70 μM from the endocardium. Tissue depth from the endocardial site is color coded on the right side of the panel.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Microscopy, Immunolabeling

    A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Stacked confocal images and reconstructed front view of optically sliced z-stack images of CX43 at a depth of 35 μm from endocardium. Gap junctions are color coded by depth and plotted within the z-stacks reconstructed from optical slices. Confocal images were acquired with a 40x oil immersion. Panel B: Stacked confocal images and reconstructed side view of optically sliced z-stack images of HCN4 + /F-actin - /(CX43) - cells (red) and HCN4 - /F-actin + /(CX43) + cells (cyan) at a depth of 40 μm and 50 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective Panel C: Optical slice shows HCN4 + /F-actin - /(CX43) - cells (red) adjacent to HCN4 - /F-actin + /(CX43) + cells (cyan). CX43 protein (green) is expressed only in cyan cells.

    Journal: bioRxiv

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    doi: 10.1101/2020.04.14.039461

    Figure Lengend Snippet: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Stacked confocal images and reconstructed front view of optically sliced z-stack images of CX43 at a depth of 35 μm from endocardium. Gap junctions are color coded by depth and plotted within the z-stacks reconstructed from optical slices. Confocal images were acquired with a 40x oil immersion. Panel B: Stacked confocal images and reconstructed side view of optically sliced z-stack images of HCN4 + /F-actin - /(CX43) - cells (red) and HCN4 - /F-actin + /(CX43) + cells (cyan) at a depth of 40 μm and 50 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective Panel C: Optical slice shows HCN4 + /F-actin - /(CX43) - cells (red) adjacent to HCN4 - /F-actin + /(CX43) + cells (cyan). CX43 protein (green) is expressed only in cyan cells.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Immunolabeling

    Panel A and B: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 36 tiled confocal images of the area of the 1350μm by 1350μm demonstrate meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. Gray color tissue was imaged in transmitted light. Panel B: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 16 tiled confocal images of the area of the 900μm by 900μm of another area of meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. CX43 is detected only in F-actin labeled cells.

    Journal: bioRxiv

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    doi: 10.1101/2020.04.14.039461

    Figure Lengend Snippet: Panel A and B: A dual immunolabeled HCN4 (red) and CX43 (green) together with F-actin labelling (cyan) merged into a single image is shown in both panels. Panel A: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 36 tiled confocal images of the area of the 1350μm by 1350μm demonstrate meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. Gray color tissue was imaged in transmitted light. Panel B: Spatial cytoarchitecture of SAN within whole mount preparations reconstructed from 16 tiled confocal images of the area of the 900μm by 900μm of another area of meshwork (red)/network(cyan) intertwining. Green dots are immunolabelled CX43 proteins. CX43 is detected only in F-actin labeled cells.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Immunolabeling, Labeling

    Panel A : An immunolabelled, whole mount image of a SAN preparation at low (2.5x) optical magnification. Panel B: Stacked confocal images and reconstructed front view of optically sliced z-stack images of HCN4 + /F-actin - cells (red) and HCN4 - /F-actin + cells (cyan) at a depth of 30 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective within ROI (yellow) shown in panel A. Panel C: Side views of the z-stack images in panel B, illustrating the intertwining cells of HCN4-meshwork and F-actin networks across the 30 μm depth.

    Journal: bioRxiv

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    doi: 10.1101/2020.04.14.039461

    Figure Lengend Snippet: Panel A : An immunolabelled, whole mount image of a SAN preparation at low (2.5x) optical magnification. Panel B: Stacked confocal images and reconstructed front view of optically sliced z-stack images of HCN4 + /F-actin - cells (red) and HCN4 - /F-actin + cells (cyan) at a depth of 30 μm from endocardium. Confocal images were acquired with a 40x oil immersion objective within ROI (yellow) shown in panel A. Panel C: Side views of the z-stack images in panel B, illustrating the intertwining cells of HCN4-meshwork and F-actin networks across the 30 μm depth.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques:

    Panel A: An Image of a whole mount SAN preparation at low (2.5x) optical magnification, demonstrating the distribution of HCN4 (red color) immunoreactive and F-actin (cyan color) labelled cells. The merged images between HCN4 and F-actin is shown in both panels. Panel B: Tiled Image of the HCN4 + /F-actin - cell meshwork (red) intertwined with the HCN4 - /F-actin + cell network (cyan) reconstructed from 4 images recorded via 10x water immersion objective within the red box in panel A.

    Journal: bioRxiv

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    doi: 10.1101/2020.04.14.039461

    Figure Lengend Snippet: Panel A: An Image of a whole mount SAN preparation at low (2.5x) optical magnification, demonstrating the distribution of HCN4 (red color) immunoreactive and F-actin (cyan color) labelled cells. The merged images between HCN4 and F-actin is shown in both panels. Panel B: Tiled Image of the HCN4 + /F-actin - cell meshwork (red) intertwined with the HCN4 - /F-actin + cell network (cyan) reconstructed from 4 images recorded via 10x water immersion objective within the red box in panel A.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques:

    Upper panels - HCN4 immunoreactive SAN cells: elongated (magenta arrows in Panel A ), novel, pyramidal-like shape cells (yellow arrow in Panels B and D ), spider-like ( Panel C ), and spindle cells ( Panel E , blue arrow). Lower panels - SAN cells loaded with Fluo-4 AM have similar shapes to immunolabelled HCN4 + cells in the upper panel. Spider-like cells ( Panel F ) are indicated by the red arrow. Novel cells with a pyramidal-like soma ( Panel G ) are indicated by yellow arrows. Spindle cells are indicated by the blue arrow ( Panel G and H ). Elongated cells ( Panel H ) are indicated by magenta arrows.

    Journal: bioRxiv

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    doi: 10.1101/2020.04.14.039461

    Figure Lengend Snippet: Upper panels - HCN4 immunoreactive SAN cells: elongated (magenta arrows in Panel A ), novel, pyramidal-like shape cells (yellow arrow in Panels B and D ), spider-like ( Panel C ), and spindle cells ( Panel E , blue arrow). Lower panels - SAN cells loaded with Fluo-4 AM have similar shapes to immunolabelled HCN4 + cells in the upper panel. Spider-like cells ( Panel F ) are indicated by the red arrow. Novel cells with a pyramidal-like soma ( Panel G ) are indicated by yellow arrows. Spindle cells are indicated by the blue arrow ( Panel G and H ). Elongated cells ( Panel H ) are indicated by magenta arrows.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques:

    Panel A: A dual immunolabeled HCN4 (red) and CX43 (green) whole mount SAN image at low optical magnification (2.5x). Merged (CX43 and HCN4) immunoreactivity is shown in all three panels. Panel B: Image within the ROI in panel A reconstructed from 4 tile images of the HCN4 + /CX43 - meshwork (red) intertwined with HCN4 - /(CX43) + network (green) taken with 10x water immersion objective. Panels C : Confocal images from the area within the ROI in panel B showing: HCN4 + cells that do not express CX43 (upper image); intertwining areas between HCN4 + /(Cx43) - meshwork (red color), and penetrating HCN4-/ CX43 + cells outlined by green dots corresponding to CX43 protein on the cell membranes (middle and lower panel). Note that HCN4 expressing cells in all three images do not express CX43.

    Journal: bioRxiv

    Article Title: Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

    doi: 10.1101/2020.04.14.039461

    Figure Lengend Snippet: Panel A: A dual immunolabeled HCN4 (red) and CX43 (green) whole mount SAN image at low optical magnification (2.5x). Merged (CX43 and HCN4) immunoreactivity is shown in all three panels. Panel B: Image within the ROI in panel A reconstructed from 4 tile images of the HCN4 + /CX43 - meshwork (red) intertwined with HCN4 - /(CX43) + network (green) taken with 10x water immersion objective. Panels C : Confocal images from the area within the ROI in panel B showing: HCN4 + cells that do not express CX43 (upper image); intertwining areas between HCN4 + /(Cx43) - meshwork (red color), and penetrating HCN4-/ CX43 + cells outlined by green dots corresponding to CX43 protein on the cell membranes (middle and lower panel). Note that HCN4 expressing cells in all three images do not express CX43.

    Article Snippet: Antibodies: HCN4+ cells were identified by rabbit polyclonal antibodies for hyperpolarization-activated, cyclic nucleotide-gate cation channels HCN4 (1:250; Alomone Labs).

    Techniques: Immunolabeling, Expressing

    The HCN4 expressions in the hippocampus of the normal and epileptic animal groups following SE. HCN4 immunoreactivities are rarely detected in all hippocampal regions, whereas its expression is observed in some interneurons (arrows in panels A2-A4). However, the immunoreactivities of HCN4 in the hippocampus following SE are unchanged depending on the time course after pilocarpine treatment (B1-D4). Bar = 280 μm (panels A1, B1, C1, and D1), 50 μm (panels A2-A4, B2-B4, C2-C4, and D2-D4). Quantitative analyses of HCN4 immunoreactivity in the normal and epileptic hippocampi following SE (E, mean ± S.E.M).

    Journal: BMB Reports

    Article Title: Alterations in hyperpolarization-activated cyclic nucleotidegated cation channel (HCN) expression in the hippocampus following pilocarpine-induced status epilepticus

    doi: 10.5483/BMBRep.2012.45.11.091

    Figure Lengend Snippet: The HCN4 expressions in the hippocampus of the normal and epileptic animal groups following SE. HCN4 immunoreactivities are rarely detected in all hippocampal regions, whereas its expression is observed in some interneurons (arrows in panels A2-A4). However, the immunoreactivities of HCN4 in the hippocampus following SE are unchanged depending on the time course after pilocarpine treatment (B1-D4). Bar = 280 μm (panels A1, B1, C1, and D1), 50 μm (panels A2-A4, B2-B4, C2-C4, and D2-D4). Quantitative analyses of HCN4 immunoreactivity in the normal and epileptic hippocampi following SE (E, mean ± S.E.M).

    Article Snippet: Sections were then incubated in below mentioned primary antibodies in PBS containing 0.3% triton X-100 overnight at room temperature; the antibodies employed were rabbit anti-HCN1, HCN2, and HCN4 IgG (Alomone labs, Israel, diluted 1:200).

    Techniques: Expressing

    The p.Pro257Ser mutant channel does not produce a measurable current and does not express on the cell membrane A, Currents were elicited from a holding current of -35 mV to a test pulse of -150 mV (fully-activated voltage) for 4 seconds and returned back to the holding current. The wild type HCN4 channel produced a current and the p.Pro257Ser mutant did not. B, confocal micrographs of wild type HCN4 and p.Pro257Ser channels expressed in CHO cells. The cells were stained with rabbit anti-HCN4 antibody (green) and DAPI (blue). The wild type HCN4 channel is expressed on the cell membrane and in the cytoplasm, whereas the p.Pro257Ser mutant channel is restricted to the cytoplasm. The scale bar denotes 50μm.

    Journal: Heart rhythm : the official journal of the Heart Rhythm Society

    Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

    doi: 10.1016/j.hrthm.2014.03.002

    Figure Lengend Snippet: The p.Pro257Ser mutant channel does not produce a measurable current and does not express on the cell membrane A, Currents were elicited from a holding current of -35 mV to a test pulse of -150 mV (fully-activated voltage) for 4 seconds and returned back to the holding current. The wild type HCN4 channel produced a current and the p.Pro257Ser mutant did not. B, confocal micrographs of wild type HCN4 and p.Pro257Ser channels expressed in CHO cells. The cells were stained with rabbit anti-HCN4 antibody (green) and DAPI (blue). The wild type HCN4 channel is expressed on the cell membrane and in the cytoplasm, whereas the p.Pro257Ser mutant channel is restricted to the cytoplasm. The scale bar denotes 50μm.

    Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

    Techniques: Mutagenesis, Produced, Staining

    Co-expression of the wild type HCN4 channel the p.Pro257Ser mutant channel produce currents that are not functionally different from wild type A, current recordings of wild type HCN4 (2 μg) and wild type HCN4 (1 μg)+p.Pro257Ser (1 μg). B, plot of current density (pA/pF) measured at -150 mV for wild type HCN4 and wild type HCN4+p.Pro257Ser. C, plots of activation curves for wild type HCN4 and wild type HCN4+p.Pro257Ser. The number in parentheses represents the number of cells. D, confocal micrographs of co-expressed wild type HCN4 and p.Pro257Ser constructs tagged with unique C-terminal epitopes in CHO cells (see methods); i) wild type HCN4-myc (green)+ wild type HCN4-V5 (red) and ii) wild type HCN4-myc (green)+p.Pro257Ser-V5(red). Co-expressed wild type HCN4-myc and wild type HCN4-V5 channels both traffick and are distributed together on cell membrane. ii) wild type HCN4-myc +p.Pro257Ser-V5 images show the wild type HCN4-myc channel expressed on the cell membrane and the p.Pro257Ser-V5 channel distributed in the cytoplasm and not on the cell membrane. Cells were also stained with DAPI (blue) to visualize the nucleus which is shown in the merged images. The scale bar denotes 50μm.

    Journal: Heart rhythm : the official journal of the Heart Rhythm Society

    Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

    doi: 10.1016/j.hrthm.2014.03.002

    Figure Lengend Snippet: Co-expression of the wild type HCN4 channel the p.Pro257Ser mutant channel produce currents that are not functionally different from wild type A, current recordings of wild type HCN4 (2 μg) and wild type HCN4 (1 μg)+p.Pro257Ser (1 μg). B, plot of current density (pA/pF) measured at -150 mV for wild type HCN4 and wild type HCN4+p.Pro257Ser. C, plots of activation curves for wild type HCN4 and wild type HCN4+p.Pro257Ser. The number in parentheses represents the number of cells. D, confocal micrographs of co-expressed wild type HCN4 and p.Pro257Ser constructs tagged with unique C-terminal epitopes in CHO cells (see methods); i) wild type HCN4-myc (green)+ wild type HCN4-V5 (red) and ii) wild type HCN4-myc (green)+p.Pro257Ser-V5(red). Co-expressed wild type HCN4-myc and wild type HCN4-V5 channels both traffick and are distributed together on cell membrane. ii) wild type HCN4-myc +p.Pro257Ser-V5 images show the wild type HCN4-myc channel expressed on the cell membrane and the p.Pro257Ser-V5 channel distributed in the cytoplasm and not on the cell membrane. Cells were also stained with DAPI (blue) to visualize the nucleus which is shown in the merged images. The scale bar denotes 50μm.

    Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

    Techniques: Expressing, Mutagenesis, Activation Assay, Construct, Staining

    ECG recordings from AF-22 who carries the HCN4 trafficking-defective p.Pro257Ser mutation Surface ECG of leads II and V1 from AF-22. AF-22 presents with the absence of p-waves indicative of AF and conduction pauses at the time of enrollment and remains in AF 12 years later.

    Journal: Heart rhythm : the official journal of the Heart Rhythm Society

    Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

    doi: 10.1016/j.hrthm.2014.03.002

    Figure Lengend Snippet: ECG recordings from AF-22 who carries the HCN4 trafficking-defective p.Pro257Ser mutation Surface ECG of leads II and V1 from AF-22. AF-22 presents with the absence of p-waves indicative of AF and conduction pauses at the time of enrollment and remains in AF 12 years later.

    Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

    Techniques: Mutagenesis

    Location of novel HCN4 coding variants in early-onset AF cases and referents A B, illustration of a HCN4 -subunit with the cytoplasmic NH2- and COOH-terminus, six transmembrane segments (S1–S6), including the S4 voltage sensor (‘+’ sign denotes amino acid residues with positive charge), the pore loop between S5 and S6, and the C-linker (CL) with the cyclic-nucleotide binding domain (CNBD). The red (seven) and blue (three) circles denote the location of the novel variants identified in the early-onset AF cases and referents, respectively.

    Journal: Heart rhythm : the official journal of the Heart Rhythm Society

    Article Title: A Novel Trafficking-defective HCN4 Mutation is Associated with Early-Onset Atrial Fibrillation

    doi: 10.1016/j.hrthm.2014.03.002

    Figure Lengend Snippet: Location of novel HCN4 coding variants in early-onset AF cases and referents A B, illustration of a HCN4 -subunit with the cytoplasmic NH2- and COOH-terminus, six transmembrane segments (S1–S6), including the S4 voltage sensor (‘+’ sign denotes amino acid residues with positive charge), the pore loop between S5 and S6, and the C-linker (CL) with the cyclic-nucleotide binding domain (CNBD). The red (seven) and blue (three) circles denote the location of the novel variants identified in the early-onset AF cases and referents, respectively.

    Article Snippet: Thirty-six hours after transfection, the cells were rinsed with PBS, fixed in 4% cold paraformaldehyde, blocked in 10% horse serum and immunolabeled with primary rabbit anti-HCN4 (Alomone labs) and Alexa546-conjugated secondary antibody (Invitrogen).

    Techniques: Binding Assay