hcn2  (Alomone Labs)


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    Structured Review

    Alomone Labs hcn2
    MTg-AMO as an improved AMO approach for miRNA target finding. ( A and B ) Western blot analysis of the protein levels of TGFBI, APC BCL2L11, <t>HCN2</t> and Cav1.2. For MTg-AMO 21/155/17 , human breast cancer MCF-7 cells were used and for MTg-AMO 1/133 , cultured neonatal rat ventricular myocytes were used. NC and NC MTg-AMO: negative control MTg-AMO. The relevant miRNA for each target mRNA is specified above the name of the target mRNA in western blot images. * P
    Hcn2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "A single anti-microRNA antisense oligodeoxyribonucleotide (AMO) targeting multiple microRNAs offers an improved approach for microRNA interference"

    Article Title: A single anti-microRNA antisense oligodeoxyribonucleotide (AMO) targeting multiple microRNAs offers an improved approach for microRNA interference

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1053

    MTg-AMO as an improved AMO approach for miRNA target finding. ( A and B ) Western blot analysis of the protein levels of TGFBI, APC BCL2L11, HCN2 and Cav1.2. For MTg-AMO 21/155/17 , human breast cancer MCF-7 cells were used and for MTg-AMO 1/133 , cultured neonatal rat ventricular myocytes were used. NC and NC MTg-AMO: negative control MTg-AMO. The relevant miRNA for each target mRNA is specified above the name of the target mRNA in western blot images. * P
    Figure Legend Snippet: MTg-AMO as an improved AMO approach for miRNA target finding. ( A and B ) Western blot analysis of the protein levels of TGFBI, APC BCL2L11, HCN2 and Cav1.2. For MTg-AMO 21/155/17 , human breast cancer MCF-7 cells were used and for MTg-AMO 1/133 , cultured neonatal rat ventricular myocytes were used. NC and NC MTg-AMO: negative control MTg-AMO. The relevant miRNA for each target mRNA is specified above the name of the target mRNA in western blot images. * P

    Techniques Used: Western Blot, Cell Culture, Negative Control

    2) Product Images from "CA2+-ACTIVATED ADENYLYL CYCLASE 1 INTRODUCES CA2+-DEPENDENCE TO BETA-ADRENERGIC STIMULATION OF HCN2 CURRENT"

    Article Title: CA2+-ACTIVATED ADENYLYL CYCLASE 1 INTRODUCES CA2+-DEPENDENCE TO BETA-ADRENERGIC STIMULATION OF HCN2 CURRENT

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2012.03.010

    Effect of over-expression of AC isoforms on HCN2 current. A . Original recordings of HCN2. The current was evoked by applying hyperpolarizing voltages from −25 to −85 mV for 5 seconds. B. Average fractional activation of measured HCN2 current. The solid lines are the fits to the Boltzmann function. AC1 activation relation differs from GFP and AC6 (p
    Figure Legend Snippet: Effect of over-expression of AC isoforms on HCN2 current. A . Original recordings of HCN2. The current was evoked by applying hyperpolarizing voltages from −25 to −85 mV for 5 seconds. B. Average fractional activation of measured HCN2 current. The solid lines are the fits to the Boltzmann function. AC1 activation relation differs from GFP and AC6 (p

    Techniques Used: Over Expression, Activation Assay

    A. Expression of recombinant adenylyl cyclase in NRVM. Cultures were co-infected with AdmHCN2 and one of the following: AdGFP, FLAG-tagged AdAC1 or FLAG-tagged AdAC6. 72 hours later cells were harvested, and the soluble membrane fraction was isolated. Soluble membranes from cultures infected with AdGFP, AdAC1 or AdAC6 were incubated with anti-FLAG or anti-HCN2 antibodies, separated by PAGE and probed with appropriate antibodies. B . Expression of endogenous HCN2 in NRVM and its co-immunoprecipitation with overexpressed adenylyl cyclases. The top panel shows detection of HCN2 in membrane fraction and the bottom panel – in anti-FLAG-immunoprecipitates.
    Figure Legend Snippet: A. Expression of recombinant adenylyl cyclase in NRVM. Cultures were co-infected with AdmHCN2 and one of the following: AdGFP, FLAG-tagged AdAC1 or FLAG-tagged AdAC6. 72 hours later cells were harvested, and the soluble membrane fraction was isolated. Soluble membranes from cultures infected with AdGFP, AdAC1 or AdAC6 were incubated with anti-FLAG or anti-HCN2 antibodies, separated by PAGE and probed with appropriate antibodies. B . Expression of endogenous HCN2 in NRVM and its co-immunoprecipitation with overexpressed adenylyl cyclases. The top panel shows detection of HCN2 in membrane fraction and the bottom panel – in anti-FLAG-immunoprecipitates.

    Techniques Used: Expressing, Recombinant, Infection, Isolation, Incubation, Polyacrylamide Gel Electrophoresis, Immunoprecipitation

    3) Product Images from "Adenosine and dopamine oppositely modulate a hyperpolarization‐activated current I h in chemosensory neurons of the rat carotid body in co‐culture"

    Article Title: Adenosine and dopamine oppositely modulate a hyperpolarization‐activated current I h in chemosensory neurons of the rat carotid body in co‐culture

    Journal: The Journal of Physiology

    doi: 10.1113/JP274743

    Localization of HCN4 and HCN2 subunits in tissue sections of rat petrosal ganglia In A , confocal immunofluorescence labelling revealed that HCN4 immunoreactivity (HCN4‐ir) is present in many petrosal neurons, and co‐localizes with several neurons expressing tyrosine hydroxylase (TH)‐ir ( B ), a marker for chemoafferent neurons; merged images are shown in C . Note co‐localization of TH‐ir and HCN4‐ir in several neurons in boxed area ( n = 3). In D , many petrosal neurons are positive for HCN2‐ir, but there is no evidence for a significant co‐localization with TH‐ir in the boxed region ( E and F ; n = 3). In control experiments omission of the primary antibody, or preincubation with blocking peptide (see Methods), resulted in the absence of all immunostaining ( n ]
    Figure Legend Snippet: Localization of HCN4 and HCN2 subunits in tissue sections of rat petrosal ganglia In A , confocal immunofluorescence labelling revealed that HCN4 immunoreactivity (HCN4‐ir) is present in many petrosal neurons, and co‐localizes with several neurons expressing tyrosine hydroxylase (TH)‐ir ( B ), a marker for chemoafferent neurons; merged images are shown in C . Note co‐localization of TH‐ir and HCN4‐ir in several neurons in boxed area ( n = 3). In D , many petrosal neurons are positive for HCN2‐ir, but there is no evidence for a significant co‐localization with TH‐ir in the boxed region ( E and F ; n = 3). In control experiments omission of the primary antibody, or preincubation with blocking peptide (see Methods), resulted in the absence of all immunostaining ( n ]

    Techniques Used: Immunofluorescence, Expressing, Marker, Blocking Assay, Immunostaining

    4) Product Images from "Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex"

    Article Title: Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex

    Journal: bioRxiv

    doi: 10.1101/2021.05.23.445332

    HCN channels are present in soma and neurite of parvalbumin-expressing basket cells in layer 5-6 of mPFC. A-C. Microscopic confocal images showing HCN1-ir ( A ), HCN2-ir ( B ), and HCN4-ir ( C ) locate in PV-ir interneuron in layer 5-6 of mPFC. Double stained with HCN channels (red) and PV (green). Arrowheads indicate double-labeled cells. Scale bar, 20 μm D. High-magnification confocal microscopy images showing that HCN1-ir localize in the soma ( d1 ) and along neurite ( d2-d3 ) of PV-ir interneuron. Silhouette frame 1 and 2 in neurite ( d1 ) is digital magnified for better view of neurite in ( d2 ) and ( d3 ), respectively. Scale bars, 20 μm in ( d1 ) and 1 μm in ( d2 ) and ( d3 ). E. High-magnification confocal microscopy images showing that HCN2-ir localize in the soma and along neurite of PV-ir interneuron. Silhouette frame in neurite (e1) is digital magnified for better view of neurite in (e2). Scale bars, 20 μm in (e1) and 1 μm in (e2).
    Figure Legend Snippet: HCN channels are present in soma and neurite of parvalbumin-expressing basket cells in layer 5-6 of mPFC. A-C. Microscopic confocal images showing HCN1-ir ( A ), HCN2-ir ( B ), and HCN4-ir ( C ) locate in PV-ir interneuron in layer 5-6 of mPFC. Double stained with HCN channels (red) and PV (green). Arrowheads indicate double-labeled cells. Scale bar, 20 μm D. High-magnification confocal microscopy images showing that HCN1-ir localize in the soma ( d1 ) and along neurite ( d2-d3 ) of PV-ir interneuron. Silhouette frame 1 and 2 in neurite ( d1 ) is digital magnified for better view of neurite in ( d2 ) and ( d3 ), respectively. Scale bars, 20 μm in ( d1 ) and 1 μm in ( d2 ) and ( d3 ). E. High-magnification confocal microscopy images showing that HCN2-ir localize in the soma and along neurite of PV-ir interneuron. Silhouette frame in neurite (e1) is digital magnified for better view of neurite in (e2). Scale bars, 20 μm in (e1) and 1 μm in (e2).

    Techniques Used: Expressing, Staining, Labeling, Confocal Microscopy

    HCN channels are present on GABAergic terminals in the mPFC. A. Low-magnification confocal images showing double stained with HCN channels (red) and GAD65 (green), a GABAergic terminal marker. The squares illustrating the cells in layer 5-6 of the mPFC. Scale bar, 40 μm B and C. Single-plane confocal images showing the HCN1-ir ( B1 ), HCN2-ir ( B2 ), HCN4-ir ( B3 ), and GAD65-ir ( C1-C3 ) at high magnification. GAD65-ir appears in punctuate structures distributed in the neuropil, as well as around unlabeled pyramidal cell soma (C1-C3). E. Merging of the paired images (B1 and C1), (B2 and C2), and (B3 and C3) shows that the puncta of GAD65-ir surround the cell bodies of HCN1-ir (B1), HCN2-ir (B2), and HCN4-ir (B3) cells. Partially overlapping areas of red (HCN) and green (GAD65) profiles showing yellow. The arrowheads indicate double-labeled cells. Scale bars represent 20 μm.
    Figure Legend Snippet: HCN channels are present on GABAergic terminals in the mPFC. A. Low-magnification confocal images showing double stained with HCN channels (red) and GAD65 (green), a GABAergic terminal marker. The squares illustrating the cells in layer 5-6 of the mPFC. Scale bar, 40 μm B and C. Single-plane confocal images showing the HCN1-ir ( B1 ), HCN2-ir ( B2 ), HCN4-ir ( B3 ), and GAD65-ir ( C1-C3 ) at high magnification. GAD65-ir appears in punctuate structures distributed in the neuropil, as well as around unlabeled pyramidal cell soma (C1-C3). E. Merging of the paired images (B1 and C1), (B2 and C2), and (B3 and C3) shows that the puncta of GAD65-ir surround the cell bodies of HCN1-ir (B1), HCN2-ir (B2), and HCN4-ir (B3) cells. Partially overlapping areas of red (HCN) and green (GAD65) profiles showing yellow. The arrowheads indicate double-labeled cells. Scale bars represent 20 μm.

    Techniques Used: Staining, Marker, Labeling

    5) Product Images from "Differential expression of hyperpolarization-activated cyclic nucleotide-gated channel subunits during hippocampal development in the mouse"

    Article Title: Differential expression of hyperpolarization-activated cyclic nucleotide-gated channel subunits during hippocampal development in the mouse

    Journal: Molecular Brain

    doi: 10.1186/s13041-015-0103-4

    Expression of HCN2 in PV-positive cells of the hippocampus at P21. (A-B, D-F, H-J, L-N, P) : The images show HCN2 labeling in different fields of the hippocampus and the DG. (A, E, I) : Note that HCN2 expression was more prominent in the sl-m and sp of the CA, particularly in CA3. Interestingly, the alveus did not show HCN2 expression at this developmental stage. In addition, the sl-m and sp of the CA3 (I) presented stronger labeling than in the CA1 (A) and the CA2 (E) . Most PV-immunopositive cells located in the sp of the CA1 (A, B, D) , CA2 (E, F, H) , CA3 (I, J, L) , and hilus of the DG (M, N, P) co-expressed HCN2. A - B , D - F , H - J , L - N , P : white arrows indicate PV-positive/HCN2-positive cells. C , G , K , O : DAPI staining. Scale bars = 20 μm.
    Figure Legend Snippet: Expression of HCN2 in PV-positive cells of the hippocampus at P21. (A-B, D-F, H-J, L-N, P) : The images show HCN2 labeling in different fields of the hippocampus and the DG. (A, E, I) : Note that HCN2 expression was more prominent in the sl-m and sp of the CA, particularly in CA3. Interestingly, the alveus did not show HCN2 expression at this developmental stage. In addition, the sl-m and sp of the CA3 (I) presented stronger labeling than in the CA1 (A) and the CA2 (E) . Most PV-immunopositive cells located in the sp of the CA1 (A, B, D) , CA2 (E, F, H) , CA3 (I, J, L) , and hilus of the DG (M, N, P) co-expressed HCN2. A - B , D - F , H - J , L - N , P : white arrows indicate PV-positive/HCN2-positive cells. C , G , K , O : DAPI staining. Scale bars = 20 μm.

    Techniques Used: Expressing, Labeling, Staining

    Expression of HCN subunits in the hippocampus at P0 and P7. (A-1) : At P0, the expression of HCN1 was strong in the alveus and so , sp , sl , and sr of the CA, and in the GCL of the dentate gyrus (DG). (E-1) : HCN2 expression at P0 was observed in the alveus , sp , sl , and sr of the CA, and in the GCL of the DG. (I-1) : HCN4 showed a similar pattern of expression to HCN1 at P0. Note that migrating cells (yellow arrows) from the ventricular zone (vz, white arrows) expressed all HCN isoforms, but the expression of HCN1 and HCN2 subunits was more prominent than that of HCN4. At P7, immunolabeling for HCN1 (A-2) , HCN2 (E-2) , and HCN4 (I-2) was observed in the sp and slm of the CA, and in the gcl of the DG. HCN1 (B, C, D) and HCN2 (F, G, H) subunits were expressed in neuronal somata, but not in astrocytes. Labeling for HCN4 was observed in the slm , as well as in the border of the slm with the molecular layer ( ml ) of the DG. (B, F, J) : Double immunofluorescence with GFAP showed that most GFAP-positive astrocytes were also immunolabeled with HCN4 in the slm, and in the border of the ml in the DG. B , C , D : yellow arrows indicate HCN1 labeling, white arrows indicate GFAP labeling. F , G , H : yellow arrows indicate HCN2 labeling. J , K , L : white arrow indicates an astrocyte double-labeled for HCN4 and GFAP. Abbreviations: GCL (or gcl), granule cell layer; sl, stratum lucidum; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bars = 20 μm.
    Figure Legend Snippet: Expression of HCN subunits in the hippocampus at P0 and P7. (A-1) : At P0, the expression of HCN1 was strong in the alveus and so , sp , sl , and sr of the CA, and in the GCL of the dentate gyrus (DG). (E-1) : HCN2 expression at P0 was observed in the alveus , sp , sl , and sr of the CA, and in the GCL of the DG. (I-1) : HCN4 showed a similar pattern of expression to HCN1 at P0. Note that migrating cells (yellow arrows) from the ventricular zone (vz, white arrows) expressed all HCN isoforms, but the expression of HCN1 and HCN2 subunits was more prominent than that of HCN4. At P7, immunolabeling for HCN1 (A-2) , HCN2 (E-2) , and HCN4 (I-2) was observed in the sp and slm of the CA, and in the gcl of the DG. HCN1 (B, C, D) and HCN2 (F, G, H) subunits were expressed in neuronal somata, but not in astrocytes. Labeling for HCN4 was observed in the slm , as well as in the border of the slm with the molecular layer ( ml ) of the DG. (B, F, J) : Double immunofluorescence with GFAP showed that most GFAP-positive astrocytes were also immunolabeled with HCN4 in the slm, and in the border of the ml in the DG. B , C , D : yellow arrows indicate HCN1 labeling, white arrows indicate GFAP labeling. F , G , H : yellow arrows indicate HCN2 labeling. J , K , L : white arrow indicates an astrocyte double-labeled for HCN4 and GFAP. Abbreviations: GCL (or gcl), granule cell layer; sl, stratum lucidum; slm, stratum lacunosum moleculare; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum. Scale bars = 20 μm.

    Techniques Used: Expressing, Immunolabeling, Labeling, Immunofluorescence

    Expression of HCN2 in PV-positive cells of the hippocampus at P56. (A-B, D, E-F, H-J, L-N, P) : Most PV-immunopositive cells located in the CA1 (A-B, D) , CA2 (E, F, H) , CA3 (I, J, L) , and DG (M, N, P) were labeled with HCN2. (A, E, I) : HCN2 expression was most prominent in the sl-m of the CA1 (A) , as well as in the sp of the CA2 (E) and CA3 (I) . Interestingly, at this stage the alveus showed HCN2 labeling, contrasting with its expression pattern at P21. A - B , D , E - F , H - J , L - N , P : white arrows indicate PV-positive/HCN2-positive cells. C , G , K , O : DAPI staining. Scale bars = 20 μm.
    Figure Legend Snippet: Expression of HCN2 in PV-positive cells of the hippocampus at P56. (A-B, D, E-F, H-J, L-N, P) : Most PV-immunopositive cells located in the CA1 (A-B, D) , CA2 (E, F, H) , CA3 (I, J, L) , and DG (M, N, P) were labeled with HCN2. (A, E, I) : HCN2 expression was most prominent in the sl-m of the CA1 (A) , as well as in the sp of the CA2 (E) and CA3 (I) . Interestingly, at this stage the alveus showed HCN2 labeling, contrasting with its expression pattern at P21. A - B , D , E - F , H - J , L - N , P : white arrows indicate PV-positive/HCN2-positive cells. C , G , K , O : DAPI staining. Scale bars = 20 μm.

    Techniques Used: Expressing, Labeling, Staining

    Expression of HCN subunits in the hippocampal region at E14.5. HCN1 (A) , HCN2 (D) , and HCN4 (G) immunolabeling was more evident in the intermediate zone (iz) than in the ventricular zone (vz). In the hippocampal iz, labeling for HCN2 and HCN4 subunits was higher than for HCN1. B , E , H : DAPI staining in the nucleus. C , F , I : merged images of HCN immunolabeling and DAPI stain. Scale bars = 20 μm.
    Figure Legend Snippet: Expression of HCN subunits in the hippocampal region at E14.5. HCN1 (A) , HCN2 (D) , and HCN4 (G) immunolabeling was more evident in the intermediate zone (iz) than in the ventricular zone (vz). In the hippocampal iz, labeling for HCN2 and HCN4 subunits was higher than for HCN1. B , E , H : DAPI staining in the nucleus. C , F , I : merged images of HCN immunolabeling and DAPI stain. Scale bars = 20 μm.

    Techniques Used: Expressing, Immunolabeling, Labeling, Staining

    Expression of HCN subunits in doublecortin (DCX)-positive cells of the dentate gyrus at P21. (A-L) : These images show our results on double immunofluorescence for HCN subunits and DCX. In the DCX-immunopositive subgranular zone we did not find labeling for HCN1 (A-D) , HCN2 (E-H) , or HCN4 (I-L) . Scale bars = 20 μm.
    Figure Legend Snippet: Expression of HCN subunits in doublecortin (DCX)-positive cells of the dentate gyrus at P21. (A-L) : These images show our results on double immunofluorescence for HCN subunits and DCX. In the DCX-immunopositive subgranular zone we did not find labeling for HCN1 (A-D) , HCN2 (E-H) , or HCN4 (I-L) . Scale bars = 20 μm.

    Techniques Used: Expressing, Immunofluorescence, Labeling

    6) Product Images from "Characterization of small fiber pathology in a mouse model of Fabry disease"

    Article Title: Characterization of small fiber pathology in a mouse model of Fabry disease

    Journal: eLife

    doi: 10.7554/eLife.39300

    Expression, function, and phenotypic reflection of hyperpolarization-activated cyclic nucleotide-gated ion channels in α-galactosidase A deficient mice. ( A ) Boxplots show the results of potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene expression in dorsal root ganglia (DRG) of young (3 months) and old (≥12 months) wildtype (WT) and α-galactosidase A deficient (GLA KO) mice. No intergroup difference was found. ( B–E ) Photomicrographs illustrate immunoreactivity of antibodies against HCN2 in DRG of young and old WT and GLA KO mice; ( F ) shows the result of quantification. Old GLA KO and WT mice showed greated HCN2 immunoreactivity compared to young littermates (p
    Figure Legend Snippet: Expression, function, and phenotypic reflection of hyperpolarization-activated cyclic nucleotide-gated ion channels in α-galactosidase A deficient mice. ( A ) Boxplots show the results of potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2) gene expression in dorsal root ganglia (DRG) of young (3 months) and old (≥12 months) wildtype (WT) and α-galactosidase A deficient (GLA KO) mice. No intergroup difference was found. ( B–E ) Photomicrographs illustrate immunoreactivity of antibodies against HCN2 in DRG of young and old WT and GLA KO mice; ( F ) shows the result of quantification. Old GLA KO and WT mice showed greated HCN2 immunoreactivity compared to young littermates (p

    Techniques Used: Expressing, Mouse Assay

    7) Product Images from "Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex"

    Article Title: Presynaptic HCN channels constrain GABAergic synaptic transmission in pyramidal cells of the medial prefrontal cortex

    Journal: bioRxiv

    doi: 10.1101/2021.05.23.445332

    HCN channels are present in soma and neurite of parvalbumin-expressing basket cells in layer 5-6 of mPFC. A-C. Microscopic confocal images showing HCN1-ir ( A ), HCN2-ir ( B ), and HCN4-ir ( C ) locate in PV-ir interneuron in layer 5-6 of mPFC. Double stained with HCN channels (red) and PV (green). Arrowheads indicate double-labeled cells. Scale bar, 20 μm D. High-magnification confocal microscopy images showing that HCN1-ir localize in the soma ( d1 ) and along neurite ( d2-d3 ) of PV-ir interneuron. Silhouette frame 1 and 2 in neurite ( d1 ) is digital magnified for better view of neurite in ( d2 ) and ( d3 ), respectively. Scale bars, 20 μm in ( d1 ) and 1 μm in ( d2 ) and ( d3 ). E. High-magnification confocal microscopy images showing that HCN2-ir localize in the soma and along neurite of PV-ir interneuron. Silhouette frame in neurite (e1) is digital magnified for better view of neurite in (e2). Scale bars, 20 μm in (e1) and 1 μm in (e2).
    Figure Legend Snippet: HCN channels are present in soma and neurite of parvalbumin-expressing basket cells in layer 5-6 of mPFC. A-C. Microscopic confocal images showing HCN1-ir ( A ), HCN2-ir ( B ), and HCN4-ir ( C ) locate in PV-ir interneuron in layer 5-6 of mPFC. Double stained with HCN channels (red) and PV (green). Arrowheads indicate double-labeled cells. Scale bar, 20 μm D. High-magnification confocal microscopy images showing that HCN1-ir localize in the soma ( d1 ) and along neurite ( d2-d3 ) of PV-ir interneuron. Silhouette frame 1 and 2 in neurite ( d1 ) is digital magnified for better view of neurite in ( d2 ) and ( d3 ), respectively. Scale bars, 20 μm in ( d1 ) and 1 μm in ( d2 ) and ( d3 ). E. High-magnification confocal microscopy images showing that HCN2-ir localize in the soma and along neurite of PV-ir interneuron. Silhouette frame in neurite (e1) is digital magnified for better view of neurite in (e2). Scale bars, 20 μm in (e1) and 1 μm in (e2).

    Techniques Used: Expressing, Staining, Labeling, Confocal Microscopy

    HCN channels are present on GABAergic terminals in the mPFC. A. Low-magnification confocal images showing double stained with HCN channels (red) and GAD65 (green), a GABAergic terminal marker. The squares illustrating the cells in layer 5-6 of the mPFC. Scale bar, 40 μm B and C. Single-plane confocal images showing the HCN1-ir ( B1 ), HCN2-ir ( B2 ), HCN4-ir ( B3 ), and GAD65-ir ( C1-C3 ) at high magnification. GAD65-ir appears in punctuate structures distributed in the neuropil, as well as around unlabeled pyramidal cell soma (C1-C3). E. Merging of the paired images (B1 and C1), (B2 and C2), and (B3 and C3) shows that the puncta of GAD65-ir surround the cell bodies of HCN1-ir (B1), HCN2-ir (B2), and HCN4-ir (B3) cells. Partially overlapping areas of red (HCN) and green (GAD65) profiles showing yellow. The arrowheads indicate double-labeled cells. Scale bars represent 20 μm.
    Figure Legend Snippet: HCN channels are present on GABAergic terminals in the mPFC. A. Low-magnification confocal images showing double stained with HCN channels (red) and GAD65 (green), a GABAergic terminal marker. The squares illustrating the cells in layer 5-6 of the mPFC. Scale bar, 40 μm B and C. Single-plane confocal images showing the HCN1-ir ( B1 ), HCN2-ir ( B2 ), HCN4-ir ( B3 ), and GAD65-ir ( C1-C3 ) at high magnification. GAD65-ir appears in punctuate structures distributed in the neuropil, as well as around unlabeled pyramidal cell soma (C1-C3). E. Merging of the paired images (B1 and C1), (B2 and C2), and (B3 and C3) shows that the puncta of GAD65-ir surround the cell bodies of HCN1-ir (B1), HCN2-ir (B2), and HCN4-ir (B3) cells. Partially overlapping areas of red (HCN) and green (GAD65) profiles showing yellow. The arrowheads indicate double-labeled cells. Scale bars represent 20 μm.

    Techniques Used: Staining, Marker, Labeling

    8) Product Images from "Differential Distribution and Function of Hyperpolarization-Activated Channels in Sensory Neurons and Mechanosensitive Fibers"

    Article Title: Differential Distribution and Function of Hyperpolarization-Activated Channels in Sensory Neurons and Mechanosensitive Fibers

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5156-03.2004

    HCN immunoreactivity in club endings of myelinated fibers accompanied by fine unmyelinated fibers. Left, HCN1 (FITC) colocalized (yellow) with peripherin (Rhodamine Red-X) in a club ending but not with fine unmyelinated fiber (arrowhead). Center, HCN2 (Rhodamine Red-X) appeared in both the fine unmyelinated fibers and in a club ending of a myelinated fiber (yellow). Right, Both club endings and fine fibers (arrowhead) labeled with FITC-NF mixture also expressed HCN4 (Rhodamine Red-X). HCN2 and HCN4 (Rhodamine Red-X) are also found in cells surrounding the fibers.
    Figure Legend Snippet: HCN immunoreactivity in club endings of myelinated fibers accompanied by fine unmyelinated fibers. Left, HCN1 (FITC) colocalized (yellow) with peripherin (Rhodamine Red-X) in a club ending but not with fine unmyelinated fiber (arrowhead). Center, HCN2 (Rhodamine Red-X) appeared in both the fine unmyelinated fibers and in a club ending of a myelinated fiber (yellow). Right, Both club endings and fine fibers (arrowhead) labeled with FITC-NF mixture also expressed HCN4 (Rhodamine Red-X). HCN2 and HCN4 (Rhodamine Red-X) are also found in cells surrounding the fibers.

    Techniques Used: Labeling

    HCN1, HCN2, and HCN4 immunoreactivity in nodose neurons. A–C , HCN immunoreactivity identified in 6–10 μm sections of nodose ganglion. HCN1 immunoreactivity was localized to a small subpopulation of neurons and, in most of these cells, was heavily localized at the plasma membrane ( A ). HCN2 ( B ) and HCN4 ( C ) immunoreactivity was present in all neurons in the ganglion. D–F , Single confocal sections through cultured nodose neurons selected for expression of HCN1 ( D ), HCN2 ( E ), and HCN4 ( F ). Heavy labeling at the membrane is again shown for HCN1 ( D ). Patches of HCN2 and HCN4 immunoreactivity were located at the cell perimeter; examples are indicated by the arrows ( E, F ). The light microscopic differential interference contrast image is also shown for each neuron. The calibration bar in C also applies to A and B , whereas calibration in F applies to E . The antibodies were preabsorbed with the immunizing peptide as shown in the figure. A control for nonspecific staining omitted the primary Ab (data not shown).
    Figure Legend Snippet: HCN1, HCN2, and HCN4 immunoreactivity in nodose neurons. A–C , HCN immunoreactivity identified in 6–10 μm sections of nodose ganglion. HCN1 immunoreactivity was localized to a small subpopulation of neurons and, in most of these cells, was heavily localized at the plasma membrane ( A ). HCN2 ( B ) and HCN4 ( C ) immunoreactivity was present in all neurons in the ganglion. D–F , Single confocal sections through cultured nodose neurons selected for expression of HCN1 ( D ), HCN2 ( E ), and HCN4 ( F ). Heavy labeling at the membrane is again shown for HCN1 ( D ). Patches of HCN2 and HCN4 immunoreactivity were located at the cell perimeter; examples are indicated by the arrows ( E, F ). The light microscopic differential interference contrast image is also shown for each neuron. The calibration bar in C also applies to A and B , whereas calibration in F applies to E . The antibodies were preabsorbed with the immunizing peptide as shown in the figure. A control for nonspecific staining omitted the primary Ab (data not shown).

    Techniques Used: Cell Culture, Expressing, Labeling, Staining

    HCN1, HCN2, HCN3, and HCN4 mRNA is expressed in nodose ganglia. PCR products resulting from the amplification of first-strand cDNA prepared with (+) or without (–) RT from nodose ganglia or brain poly A+ RNA with HCN1-, HCN2-, HCN3-, and HCN4-specific oligonucleotides were separated by electrophoresis and transferred to nylon membranes (Ambion). After Southern hybridization with 32 P-labeled specific internal oligomers, the autoradiogram showed a positive signal for all four channels from nodose and rat brain in the (+) RT lanes and no signals in the control (–) RT. The oligonucleotide probes amplify cDNA of 641 bp for HCN1, 638 bp for HCN2, 509 bp for HCN3, and 635 bp for HCN4.
    Figure Legend Snippet: HCN1, HCN2, HCN3, and HCN4 mRNA is expressed in nodose ganglia. PCR products resulting from the amplification of first-strand cDNA prepared with (+) or without (–) RT from nodose ganglia or brain poly A+ RNA with HCN1-, HCN2-, HCN3-, and HCN4-specific oligonucleotides were separated by electrophoresis and transferred to nylon membranes (Ambion). After Southern hybridization with 32 P-labeled specific internal oligomers, the autoradiogram showed a positive signal for all four channels from nodose and rat brain in the (+) RT lanes and no signals in the control (–) RT. The oligonucleotide probes amplify cDNA of 641 bp for HCN1, 638 bp for HCN2, 509 bp for HCN3, and 635 bp for HCN4.

    Techniques Used: Polymerase Chain Reaction, Amplification, Electrophoresis, Hybridization, Labeling

    HCN immunoreactivity in aortic baroreceptor terminals of myelinated fibers. Top, A collapsed Z-series stack of 0.4 μm confocal sections through a bush baroreceptor terminal shows localization of HCN1 (left) and PGP9.5 (right). PGP9.5 is a ubiquitin hydrolase expressed in neuronal–neuroendocrine cells. Middle, Bush ending is colabeled with HCN2 on the left and the neurofilament mixture on the right. Bottom, HCN4 immunoreactivity on the left is localized to the bush ending identified using the neurofilament mixture (right).
    Figure Legend Snippet: HCN immunoreactivity in aortic baroreceptor terminals of myelinated fibers. Top, A collapsed Z-series stack of 0.4 μm confocal sections through a bush baroreceptor terminal shows localization of HCN1 (left) and PGP9.5 (right). PGP9.5 is a ubiquitin hydrolase expressed in neuronal–neuroendocrine cells. Middle, Bush ending is colabeled with HCN2 on the left and the neurofilament mixture on the right. Bottom, HCN4 immunoreactivity on the left is localized to the bush ending identified using the neurofilament mixture (right).

    Techniques Used:

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    Alomone Labs hcn2
    MTg-AMO as an improved AMO approach for miRNA target finding. ( A and B ) Western blot analysis of the protein levels of TGFBI, APC BCL2L11, <t>HCN2</t> and Cav1.2. For MTg-AMO 21/155/17 , human breast cancer MCF-7 cells were used and for MTg-AMO 1/133 , cultured neonatal rat ventricular myocytes were used. NC and NC MTg-AMO: negative control MTg-AMO. The relevant miRNA for each target mRNA is specified above the name of the target mRNA in western blot images. * P
    Hcn2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti hcn2 antibody
    Effect of chronic training on HCN1, KCN2 and HCN4 protein expression determined by immunocytochemical techniques in sedentary and trained dog. Bar diagram on top left indicates that the relative density of dog cardiomyocytes with HCN4 immunolabelling obtained from the trained group (n=30 cells/6 dogs) is significantly increased compared to that measured in the sedentary group (n=30 cells/6 dogs). Original immunofluorescent images are shown on the left . Bottom panels indicate lack of effect of chronic training on the relative density of dog cardiomyocytes with HCN1 and <t>HCN2</t> immunolabelling (n=30 – 30 cells/6 – 6 dogs for sedentary and trained groups, respectively). Figure 7–Source Data 1 Effect of chronic training on HCN4 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 2 Effect of chronic training on HCN1 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 3 Effect of chronic training on HCN2 protein expression determined by immunocytochemical technique in sedentary and trained dogs.
    Anti Hcn2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MTg-AMO as an improved AMO approach for miRNA target finding. ( A and B ) Western blot analysis of the protein levels of TGFBI, APC BCL2L11, HCN2 and Cav1.2. For MTg-AMO 21/155/17 , human breast cancer MCF-7 cells were used and for MTg-AMO 1/133 , cultured neonatal rat ventricular myocytes were used. NC and NC MTg-AMO: negative control MTg-AMO. The relevant miRNA for each target mRNA is specified above the name of the target mRNA in western blot images. * P

    Journal: Nucleic Acids Research

    Article Title: A single anti-microRNA antisense oligodeoxyribonucleotide (AMO) targeting multiple microRNAs offers an improved approach for microRNA interference

    doi: 10.1093/nar/gkn1053

    Figure Lengend Snippet: MTg-AMO as an improved AMO approach for miRNA target finding. ( A and B ) Western blot analysis of the protein levels of TGFBI, APC BCL2L11, HCN2 and Cav1.2. For MTg-AMO 21/155/17 , human breast cancer MCF-7 cells were used and for MTg-AMO 1/133 , cultured neonatal rat ventricular myocytes were used. NC and NC MTg-AMO: negative control MTg-AMO. The relevant miRNA for each target mRNA is specified above the name of the target mRNA in western blot images. * P

    Article Snippet: The sample was incubated overnight at 4°C with primary antibodies for TGFBI, APC and BCL2L11 (Santa Cruz), and for HCN2 and Cav1.2 (Alomone Labs).

    Techniques: Western Blot, Cell Culture, Negative Control

    Domains mediating the interaction of VAPB with HCN2. A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: Domains mediating the interaction of VAPB with HCN2. A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Construct, Expressing, Chemiluminescence Immunoassay

    VAPB determines surface expression and dendritic localization of HCN2. A ) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope ( EGFP HCN2 HA Ex ) alone or cotransfected with VAPB or the TM segment of VAPB (TM VAPB ). B ) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFP HCN2 HA Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 prote in expression. C ) Chemiluminescence surface expression assay as in B , but using TM VAPB (1.6 ± 0.1). D ) Immunocytochemistry of HA VAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with HA VAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E ) Immunocytochemistry experiment as in D , but transfecting the ALS8 mutation HA VAPB P56S (magenta), leading to an aggregation of VAPB P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm ( A , D , E ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. ** P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: VAPB determines surface expression and dendritic localization of HCN2. A ) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope ( EGFP HCN2 HA Ex ) alone or cotransfected with VAPB or the TM segment of VAPB (TM VAPB ). B ) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFP HCN2 HA Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 prote in expression. C ) Chemiluminescence surface expression assay as in B , but using TM VAPB (1.6 ± 0.1). D ) Immunocytochemistry of HA VAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with HA VAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E ) Immunocytochemistry experiment as in D , but transfecting the ALS8 mutation HA VAPB P56S (magenta), leading to an aggregation of VAPB P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm ( A , D , E ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. ** P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Expressing, Live Cell Imaging, Transfection, Cotransfection, Western Blot, Immunocytochemistry, Staining, Mutagenesis, Fluorescence

    Codistribution of VAPs with HCN2 and contribution to thalamic I h . A – E ), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas ( A ), hippocampus ( B ), thalamus ( C ), cerebellum ( D ) (arrows point to interneurons in the granular layer), and spinal cord ( E ). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F ) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB −/− mice. G ) The I h current was significantly reduced in VAPB −/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H ) Average activation curves of the VB I h current for control and VAPB −/− mice. V 1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB −/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm ( A–C , E ), 100 µm ( D ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: Codistribution of VAPs with HCN2 and contribution to thalamic I h . A – E ), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas ( A ), hippocampus ( B ), thalamus ( C ), cerebellum ( D ) (arrows point to interneurons in the granular layer), and spinal cord ( E ). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F ) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB −/− mice. G ) The I h current was significantly reduced in VAPB −/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H ) Average activation curves of the VB I h current for control and VAPB −/− mice. V 1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB −/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm ( A–C , E ), 100 µm ( D ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: In Situ Hybridization, Labeling, Expressing, Patch Clamp, Mouse Assay, Activation Assay

    VAPB selectively increases HCN1 and HCN2 currents. A ) Y2H direct interaction assay. Transformation control (-LW), leucine, and tryptophan dropout. Interaction read-out (-LWHA), additional dropout of histidine and adenine. pAL-Alg5, positive control. pPR3-N, negative control. B ) Topology of VAPB. C ) GST VAPB pull-down of HCN2 EGFP using transfected HeLa cells. D ) GST VAPA, GST VAMP1, or GST VAMP2 pull-down of HCN2 EGFP using transfected HeLa cells. E ) GST VAPA pull-down of HCN2 and endogenous VAPB, using HCN2 EGFP transfected HeLa cells. F ) Pull-down of in vitro translated HCN2 (untagged). G ) Pull-down of HCN2 from rat brain lysates. H , I ) Representative currents ( H ) of HCN2 expressed in oocytes alone or with VAPB and the relative current amplitudes ( I ) analyzed over 3 d. J ) Relative currents of HCN1, HCN2, and HCN4 alone or coexpressed with VAPB. K , L ) Relative currents of different potassium channels ( K ) coexpressed with VAPB and of HCN2 ( L ) coexpressed with VAPA, VAPB, or VAPC. M ) Relative currents of HCN2 coexpressed with a mixture of VAPA/B (1:1). N ) Representative macropatch recordings in different configurations: on cell (o.c.), inside-out after patch excision (i.o.), and after application of 100 µM cAMP (i.o.+100 µM cAMP). O , P ) Activation curves for HCN2 alone ( n = 6) ( O ), recorded as in N , or after coexpression with VAPB ( n = 8) ( P ). Q ) V 1/2 values for HCN2 expressed alone or with VAPB in different patch modes. R ) Relative currents of HCN2 HA Ex alone or with VAPB. S ) Relative surface expression of HCN2 HA Ex expressed alone or with VAPB, analyzed as relative light units (RLUs). T ) Relative currents of HCN2 expressed alone or with VAPB or VAPB P56S . All data are presented as means ± sem . The number of cells ( n ) is indicated in the bar graphs. N.s., not significant. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: VAPB selectively increases HCN1 and HCN2 currents. A ) Y2H direct interaction assay. Transformation control (-LW), leucine, and tryptophan dropout. Interaction read-out (-LWHA), additional dropout of histidine and adenine. pAL-Alg5, positive control. pPR3-N, negative control. B ) Topology of VAPB. C ) GST VAPB pull-down of HCN2 EGFP using transfected HeLa cells. D ) GST VAPA, GST VAMP1, or GST VAMP2 pull-down of HCN2 EGFP using transfected HeLa cells. E ) GST VAPA pull-down of HCN2 and endogenous VAPB, using HCN2 EGFP transfected HeLa cells. F ) Pull-down of in vitro translated HCN2 (untagged). G ) Pull-down of HCN2 from rat brain lysates. H , I ) Representative currents ( H ) of HCN2 expressed in oocytes alone or with VAPB and the relative current amplitudes ( I ) analyzed over 3 d. J ) Relative currents of HCN1, HCN2, and HCN4 alone or coexpressed with VAPB. K , L ) Relative currents of different potassium channels ( K ) coexpressed with VAPB and of HCN2 ( L ) coexpressed with VAPA, VAPB, or VAPC. M ) Relative currents of HCN2 coexpressed with a mixture of VAPA/B (1:1). N ) Representative macropatch recordings in different configurations: on cell (o.c.), inside-out after patch excision (i.o.), and after application of 100 µM cAMP (i.o.+100 µM cAMP). O , P ) Activation curves for HCN2 alone ( n = 6) ( O ), recorded as in N , or after coexpression with VAPB ( n = 8) ( P ). Q ) V 1/2 values for HCN2 expressed alone or with VAPB in different patch modes. R ) Relative currents of HCN2 HA Ex alone or with VAPB. S ) Relative surface expression of HCN2 HA Ex expressed alone or with VAPB, analyzed as relative light units (RLUs). T ) Relative currents of HCN2 expressed alone or with VAPB or VAPB P56S . All data are presented as means ± sem . The number of cells ( n ) is indicated in the bar graphs. N.s., not significant. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Transformation Assay, Positive Control, Negative Control, Transfection, In Vitro, Activation Assay, Expressing

    Effect of over-expression of AC isoforms on HCN2 current. A . Original recordings of HCN2. The current was evoked by applying hyperpolarizing voltages from −25 to −85 mV for 5 seconds. B. Average fractional activation of measured HCN2 current. The solid lines are the fits to the Boltzmann function. AC1 activation relation differs from GFP and AC6 (p

    Journal: Journal of molecular and cellular cardiology

    Article Title: CA2+-ACTIVATED ADENYLYL CYCLASE 1 INTRODUCES CA2+-DEPENDENCE TO BETA-ADRENERGIC STIMULATION OF HCN2 CURRENT

    doi: 10.1016/j.yjmcc.2012.03.010

    Figure Lengend Snippet: Effect of over-expression of AC isoforms on HCN2 current. A . Original recordings of HCN2. The current was evoked by applying hyperpolarizing voltages from −25 to −85 mV for 5 seconds. B. Average fractional activation of measured HCN2 current. The solid lines are the fits to the Boltzmann function. AC1 activation relation differs from GFP and AC6 (p

    Article Snippet: To explore the possibility of direct interaction with HCN2 channels, we immunoprecipitated AC with anti-FLAG antibodies, separated the obtained proteins by PAGE and probed with anti-HCN2 antibodies ( ; middle panel).

    Techniques: Over Expression, Activation Assay

    A. Expression of recombinant adenylyl cyclase in NRVM. Cultures were co-infected with AdmHCN2 and one of the following: AdGFP, FLAG-tagged AdAC1 or FLAG-tagged AdAC6. 72 hours later cells were harvested, and the soluble membrane fraction was isolated. Soluble membranes from cultures infected with AdGFP, AdAC1 or AdAC6 were incubated with anti-FLAG or anti-HCN2 antibodies, separated by PAGE and probed with appropriate antibodies. B . Expression of endogenous HCN2 in NRVM and its co-immunoprecipitation with overexpressed adenylyl cyclases. The top panel shows detection of HCN2 in membrane fraction and the bottom panel – in anti-FLAG-immunoprecipitates.

    Journal: Journal of molecular and cellular cardiology

    Article Title: CA2+-ACTIVATED ADENYLYL CYCLASE 1 INTRODUCES CA2+-DEPENDENCE TO BETA-ADRENERGIC STIMULATION OF HCN2 CURRENT

    doi: 10.1016/j.yjmcc.2012.03.010

    Figure Lengend Snippet: A. Expression of recombinant adenylyl cyclase in NRVM. Cultures were co-infected with AdmHCN2 and one of the following: AdGFP, FLAG-tagged AdAC1 or FLAG-tagged AdAC6. 72 hours later cells were harvested, and the soluble membrane fraction was isolated. Soluble membranes from cultures infected with AdGFP, AdAC1 or AdAC6 were incubated with anti-FLAG or anti-HCN2 antibodies, separated by PAGE and probed with appropriate antibodies. B . Expression of endogenous HCN2 in NRVM and its co-immunoprecipitation with overexpressed adenylyl cyclases. The top panel shows detection of HCN2 in membrane fraction and the bottom panel – in anti-FLAG-immunoprecipitates.

    Article Snippet: To explore the possibility of direct interaction with HCN2 channels, we immunoprecipitated AC with anti-FLAG antibodies, separated the obtained proteins by PAGE and probed with anti-HCN2 antibodies ( ; middle panel).

    Techniques: Expressing, Recombinant, Infection, Isolation, Incubation, Polyacrylamide Gel Electrophoresis, Immunoprecipitation

    Effect of chronic training on HCN1, KCN2 and HCN4 protein expression determined by immunocytochemical techniques in sedentary and trained dog. Bar diagram on top left indicates that the relative density of dog cardiomyocytes with HCN4 immunolabelling obtained from the trained group (n=30 cells/6 dogs) is significantly increased compared to that measured in the sedentary group (n=30 cells/6 dogs). Original immunofluorescent images are shown on the left . Bottom panels indicate lack of effect of chronic training on the relative density of dog cardiomyocytes with HCN1 and HCN2 immunolabelling (n=30 – 30 cells/6 – 6 dogs for sedentary and trained groups, respectively). Figure 7–Source Data 1 Effect of chronic training on HCN4 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 2 Effect of chronic training on HCN1 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 3 Effect of chronic training on HCN2 protein expression determined by immunocytochemical technique in sedentary and trained dogs.

    Journal: bioRxiv

    Article Title: Cardiac electrophysiological remodeling associated with enhanced arrhythmia susceptibilty in a canine model of elite exercise

    doi: 10.1101/2022.07.13.499876

    Figure Lengend Snippet: Effect of chronic training on HCN1, KCN2 and HCN4 protein expression determined by immunocytochemical techniques in sedentary and trained dog. Bar diagram on top left indicates that the relative density of dog cardiomyocytes with HCN4 immunolabelling obtained from the trained group (n=30 cells/6 dogs) is significantly increased compared to that measured in the sedentary group (n=30 cells/6 dogs). Original immunofluorescent images are shown on the left . Bottom panels indicate lack of effect of chronic training on the relative density of dog cardiomyocytes with HCN1 and HCN2 immunolabelling (n=30 – 30 cells/6 – 6 dogs for sedentary and trained groups, respectively). Figure 7–Source Data 1 Effect of chronic training on HCN4 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 2 Effect of chronic training on HCN1 protein expression determined by immunocytochemical technique in sedentary and trained dogs. Figure 7–Source Data 3 Effect of chronic training on HCN2 protein expression determined by immunocytochemical technique in sedentary and trained dogs.

    Article Snippet: After the incubation period, cells were labelled overnight at 4°C with anti-KChIP2 (Alomone, #APC-142, RRID:AB_2756744), anti-Kv4.3 (Alomone, #APC-017, RRID:AB_2040178), anti-HCN1 (Alomone, #APC-056, RRID:AB_2039900), anti-HCN2 (Alomone, #APC-030, RRID:AB_2313726) and anti-HCN4 (Alomone, #APC-052, RRID:AB_2039906) primary antibody diluted to 1:50.

    Techniques: Expressing