hcn4  (Alomone Labs)

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

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

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

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

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

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

Journal: The Journal of Physiology

Article Title: Hyperpolarisation-activated cyclic nucleotide-gated channels regulate the spontaneous firing rate of olfactory receptor neurons and affect glomerular formation in mice

doi: 10.1113/jphysiol.2012.247361

Figure Lengend Snippet: HCN4 over-expression resulted in reductions in olfactory glomeruli

Article Snippet: The following primary antibodies were used in the present study: anti-HCN1 (1:1000, Dr Shigemoto, National Institute for Physiological Sciences, Japan), anti-HCN2, anti-HCN3, anti-HCN4 (1:500, Alomone Labs, Jerusalem, Israel) and anti-olfactory marker protein (OMP, 1:400, Wako, Japan).

Techniques: Over Expression

Journal: The Journal of Physiology

Article Title: Hyperpolarisation-activated cyclic nucleotide-gated channels regulate the spontaneous firing rate of olfactory receptor neurons and affect glomerular formation in mice

doi: 10.1113/jphysiol.2012.247361

Figure Lengend Snippet: HCN4 channel over-expression boosts the rate of spontaneous ORN firing activity

Article Snippet: The following primary antibodies were used in the present study: anti-HCN1 (1:1000, Dr Shigemoto, National Institute for Physiological Sciences, Japan), anti-HCN2, anti-HCN3, anti-HCN4 (1:500, Alomone Labs, Jerusalem, Israel) and anti-olfactory marker protein (OMP, 1:400, Wako, Japan).

Techniques: Over Expression, Activity Assay

Journal: The Journal of Physiology

Article Title: Hyperpolarisation-activated cyclic nucleotide-gated channels regulate the spontaneous firing rate of olfactory receptor neurons and affect glomerular formation in mice

doi: 10.1113/jphysiol.2012.247361

Figure Lengend Snippet: HCN2 and HCN4 are predominant in ORNs

Article Snippet: The following primary antibodies were used in the present study: anti-HCN1 (1:1000, Dr Shigemoto, National Institute for Physiological Sciences, Japan), anti-HCN2, anti-HCN3, anti-HCN4 (1:500, Alomone Labs, Jerusalem, Israel) and anti-olfactory marker protein (OMP, 1:400, Wako, Japan).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of FPMINT analogues (listed in Supplementary Table S2 ) on [ 3 H]uridine uptake by ENT1 and ENT2 [ 3 H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in Supplementary Table S2 (10 nM–100 μM).

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of FPMINT analogues (listed in Supplementary Table S1 ) on [ 3 H]uridine uptake by ENT1 and ENT2 [ 3 H]uridine uptake (1 μM, 2 μCi/ml) in (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells was measured in the presence of various concentrations of FPMINT analogues listed in Supplementary Table S1 (10 nM–100 μM).

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Effects of compound 3c on cytotoxicity and protein expressions of ENT1 and ENT2. Cytotoxicity of compound 3c was measured after 24 h or 48 h treatment of PK15NTD/ENT1 and PK15NTD/ENT2 cells with various concentrations of compound 3c (0.5, 5 and 50 μM) or a vehicle (0.5% DMSO, as a control) (A) The cell viability and (B) cell membrane integrity were determined by the MTT assay and LDH release, respectively (C) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. Western blotting assay was performed to determine the protein expression levels of ENT1 and ENT2, with β-actin as an internal reference. Cell surface proteins of (E) PK15NTD/ENT1 and (F) PK15NTD/ENT2 cells were biotinylated and treated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for 24 or 48 h. After cleavage of extracellular biotin, internalized biotinylated proteins were precipitated with immobilized streptavidin beads and detected with an anti-ENT1 and anti-ENT2 antibodies by western blotting. Total ENT1 and ENT2 from whole-cell lysates were also detected for a comparison. β-actin served as an internal reference. Representative blots are from three independent experiments. Data are presented as mean ± SE of three experiments.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: MTT Assay, Incubation, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Competitive inhibition of ENT1 and ENT2 by compound 3c. Kinetic study of [ 3 H]uridine uptake (0.1 μM–1 mM) was measured in (A) PK15NTD/ENT1 and (D) PK15NTD/ENT2 cells in the presence of various concentrations of compound 3c (0, 0.01, 0.1, 1 and 10 µM) (B,E) 1/ V versus 1/[S] plots of each curve in (A) and (D) , respectively (C,F) The plot of slopes of each line in (B) and (E) , respectively, versus the concentration of compound 3c . Data are presented as mean ± SE of three experiments.

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: Inhibition, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Structure-Activity Relationship Studies of 4-((4-(2-fluorophenyl)piperazin-1-yl)methyl)-6-imino-N-(naphthalen-2-yl)-1,3,5-triazin-2-amine (FPMINT) Analogues as Inhibitors of Human Equilibrative Nucleoside Transporters

doi: 10.3389/fphar.2022.837555

Figure Lengend Snippet: Reversibility of the inhibitory effects of compound 3c on ENT1 and ENT2 (A) PK15NTD/ENT1 and (B) PK15NTD/ENT2 cells were incubated with 50 μM of compound 3c or a vehicle (0.5% DMSO, as a control) for different time (0–60 min) The cells were then washed five times. After wash-out for every single time [ 3 H]uridine uptake (1 μM, 2 μCi/ml) was measured. Data are presented as mean ± SE of three experiments. * p

Article Snippet: The membranes were blocked with 5% (w/v) non-fat dry milk in PBS for 1 h at room temperature and then incubated with rabbit polyclonal anti-ENT1 or anti-ENT2 primary antibodies (Alomone Labs, Jerusalem, Israel) (1:200 [v/v] dilution in a blocking solution) at 4°C overnight.

Techniques: Incubation