polyclonal rabbit α hcn2  (Alomone Labs)

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

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

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

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

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

Journal: Science (New York, N.Y.)

Article Title: Astrocyte Ca2+-evoked ATP release regulates myelinated axon excitability and conduction speed *

doi: 10.1126/science.abh2858

Figure Lengend Snippet: ATP release from astrocytes may target adenosine receptors on myelinated axons of layer V pyramidal neurons. ( A ) Using luciferin-luciferase to detect ATP puffed into extracellular solution. ( B ) Response to 2-photon excitation uncaging Ca 2+ in astrocytes evoked a luciferin-luciferase signal, unless the excitation failed to raise astrocyte [Ca 2+ ]i (No Ca 2+ u.; see Fig. S2 ). ( C ) Quantification of experiments in B in 7 cells. ( D-H ) A 2a Rs are present in the AIS ( D ) where they overlap with voltage-gated Na + channel (Na v ) expression ( E , mean of 14 Na v and 25 A 2a R profiles) and at the node of Ranvier ( F ) where they overlap with Na v and are flanked by Caspr labeling ( G , mean of 48 Caspr, 48 A 2a R and 24 Na v profiles). ( H ) Percentage of 25 AISs and 134 nodes that express A 2a Rs. ( I-M ) HCN2 channel subunits are present in the AIS ( I ) where they overlap with voltage-gated Na + channel (Na v ) expression ( J , mean of 19 Na V and 16 HCN2 profiles) and at the node of Ranvier ( K ) where they overlap with Na v and are flanked by Caspr labeling ( L , mean of 53 Caspr and HCN2 profiles). ( M ) Percentage of 49 AISs and 98 nodes that express HCN2. Panels A-E and I-J are on rats; F-G and K-L are on mouse; H and M combine rat AIS and mouse node data.

Article Snippet: The slices were permeabilized with 0.2% Triton X-100 (Sigma) in a blocking solution (10% goat serum in 0.01 M PBS) for 1 h. The slices were then incubated overnight at 4 °C with the following primary antibodies, as required: rabbit anti-A2a R (Abcam, ab3461, 1:100), mouse anti-A2a R (Millipore, 05-717 which has been validated as giving no labelling in A2a R KO tissue , 1:200), rabbit anti-A1 R (Abcam, ab82477, 1:100), rabbit anti-A2bR (Cohesion, CPA3755, 1:100), mouse anti-Ankyrin G (Neuromab, N106/36, 1:500), mouse anti-panNav (Sigma, K58/35, 1:100), mouse anti-Caspr (Neuromab, K65/35, 1:100), chicken anti-GFP (Millipore, AB16901, 1:1000), rabbit anti-GFAP (Millipore, AB5804, 1:500), rabbit anti-HCN1 (Alomone, APC 056, 1:100) and rabbit anti-HCN2 (Alomone, APC 030, 1:100).

Techniques: Luciferase, Expressing, Labeling

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