Journal: Journal of neurophysiology

Article Title: The HCN4 channel modulates external tufted cell burst firing

doi: 10.1152/jn.00194.2026

Figure Lengend Snippet: Effect of intracellular delivery of HCN blocking antisera on external tufted cell firing. A : External tufted cell firing 1 minute after whole cell configuration baseline ( top ). After 6 min intracellular diffusion with HCN4 blocking antisera external tufted cell spiking was terminated in this cell ( bottom ); representative biocytin filled external tufted cell shown. B : External tufted cell firing 1 minute after whole cell configuration baseline ( top ). After 6 min (shown here, to longer time 30 min recorded in this cell) intracellular diffusion with HCN2 blocking antisera external tufted cell spiking was unaffected ( bottom ). C : Group data showing firing rates with blocking antisera represented as a percent of the pre-equilibration baseline.

Article Snippet: Membrane blots were incubated in TBST buffer with 5% powder milk for 1 h, and then probed in 4 °C overnight with one of the following primary antibodies in same buffer: rabbit anti-HCN1 (#APC-056, 1:200), -HCN2 (#APC-030, 1:200), -HCN3 (#APC-057, 1:200) and -HCN4 (#APC-052, 1:200) antibody (Alomone Labs, Jerusalem, Israel) and rabbit anti-β-actin (1:2500; Cell Signaling Technology, Danvers, MA, USA).

Techniques: Blocking Assay, Diffusion-based Assay

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: Disease-free survival curve and overall survival curve by the methylation level in the proximal region of HCN2 promoter. No statistical significance was found for each curve by the log-rank test (P=0.78 or P=0.56).

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Methylation

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: The schematic illustration by MethPrimer of the CpG islands and CpG dinucleotides in the HCN2 proximal promoter (2650 bp), 5′-UTR exon (53 bp to the start codon, ATG), and first CDS exon (632 bp including ATG). The total sequence length: 4685 bp. The island 1 (far left) is 140 bp (1615-1754); island 2 is 162 bp (2081-2242); the island 3 is 112 bp (2466-2577), the island 4 is 167 bp (2647-2813), and the island 5 (far right) that is composed of 5′-UTR exon, first CDS exon, and the proximal part of the first intron is 2290 bp (2202-4491). F4 and R4 are the primers used for bisulfite sequencing (BS-seq).

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Sequencing, Methylation Sequencing

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: The schematic illustration of the regions in the proximal HCN2 promoter that are validated by bisulfite sequencing (BS-seq) and qMSP. BS-seq-F, bisulfite sequencing forward primer; BS-seq-R, bisulfite sequencing reverse primer; qMSP-F, qMSP forward primer; qMSP-R, qMSP reverse primer; qMSP-Probe, qMSP probe. All numberings are in reference to the start codon, ATG.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Methylation Sequencing

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: Top 32 genes detected by methylation array.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Methylation

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: The schematic illustration of 10 CpG dinucleotides in the proximal region of HCN2 promoter and their methylation status validated by bisulfite sequencing in a small validation cohort of 6 normal and 6 ACC tumor samples.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Methylation, Methylation Sequencing, Biomarker Discovery

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: The correlation of the clinical variables and patient outcomes with the median HCN2 promoter methylation status in a proximal region detected by the methylation array.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Methylation, Control, Dissection, Translocation Assay

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: qMSP validation of the differential methylation status in the proximal region of HCN2 promoter detected by methylation array. qMSP validation was done between control (n=20) and ACC (n=32) cases in a separate, large FFPE cohort. P=0.04 between control and ACC cases.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Biomarker Discovery, Methylation, Control

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: HCN2 methylation status is correlated with any recurrence (regional, local or distant) after primary treatment in ACC cases. ‘No’ means ACC cases that had no recurrence after primary treatment (n=16). ‘Yes’ means ACC cases that had any recurrence after primary treatment (n=15). P=0.02 by the non-parametric Wilcoxon rank-sum test between ‘No’ and ‘Yes’.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Methylation

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: Survival by promoter methylation level of HCN2 gene among ACC cases.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Methylation

Journal: International Journal of Oncology

Article Title: Identification of methylated genes in salivary gland adenoid cystic carcinoma xenografts using global demethylation and methylation microarray screening

doi: 10.3892/ijo.2016.3532

Figure Lengend Snippet: HCN2 IHC analysis between normal control and ACC cases.

Article Snippet: For IHC, a mouse monoclonal HCN2 antibody (S71-37) was obtained from Novus Biologicals LLC (Littleton, CO, USA).

Techniques: Control, Staining

Journal: Cell reports

Article Title: Optogenetic Activation of Striatopallidal Neurons Reveals Altered HCN Gating in DYT1 Dystonia.

doi: 10.1016/j.celrep.2020.107644

Figure Lengend Snippet: Figure 7. ConcanamycinA Treatment Modulates HCN1 and HCN2 Expression (A) Cartoon showing the mechanism of concanamycinA (concan) treatment upon vesicular release. (B) sIPSCs recorded in slices of Tor1a+/+ and Tor1a+/DGAG treated with saline (black traces) or with concan (red traces). (C) Boxplot summarizes the effect of concan on firing frequency in Tor1a+/+ and Tor1a+/DGAG. (D) Western blots showing immunoreactivity of HCN1 (untreated N = 5, treated N = 7) and HCN2 (untreated N = 6, treated N = 6) subunits from GPe extracts from slices of animals treated with saline or concan. Protein values were normalized to the corresponding expression of tubulin and actin, respectively. Bar plots illustrate the effect of treatment on HCN subunits level in Tor1a+/+ and Tor1a+/DGAG mice. Levels were normalized to the control (CTRL) level of Tor1a+/+. (E) Cell-attached recordings of autonomous pacemaking activity of GP neurons from Tor1a+/DGAG treated with control solution (black) or concan (red). Boxplots show effect of concan on firing frequency and CV. (F) Whole-cell recordings from GP neurons of Tor1a+/DGAG during optical striatal stimulation (blue lines, dual pulse, 20 Hz) after treatment with concan. Five superimposed traces were aligned on the striatal stimulation. Peri-stimulus time histogram (PSTH) shows firing pause and resetting phase triggered by striatal stimulation. Each dot represents single measurements.

Article Snippet: Protein extracts were stored at 80 C and 10 mg of each sample, in Page LDS sample buffer 4x (Invitrogen) was analyzed to detect: TA (1:1000, Abcam AB_2240792), PV (1:2000, Abcam AB_956206), GAD67 (1:2000, Sigma Aldrich AB_261978), GAD65 (1:1000, Sigma Aldrich AB_10762670), VGAT (1:1000 SySy AB_887871), Gephyrin (1:500, Proteintech AB_2247770), HCN2 (1:1000, Proteintech AB_10951108) HCN1 (1:1000, Proteintech AB_11182929), rabbit anti-D2R (1:1000, Millipore AB_2094980), antiactin (Sigma Aldrich AB_476744) as loading control.

Techniques: Expressing, Saline, Western Blot, Control, Activity Assay

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Representative traces of HCN currents elicited by –150 mV in NAc ChIs from WT and cKO mice before (black, Ctrl) and after DA (1 μM, red). (B) Histograms showing the amplitude of HCN currents at -150 mV in control or DA-treated ChIs from NAc in WT (n = 8 neurons/ 4 mice) and cKO (n = 4 neurons/ 3 mice). Two-way RM ANOVA. Genotype X Treatment F [ , ] = 3.39; P= 0.09. Genotype F [ , ] = 5.90; P= 0.03. Treatment F [ , ] = 4.48; P= 0.06 * p < 0.05 by Bonferroni (C) Effect by aCSF without or with neostigmine (500 nM, arrow) on NAc ACh efflux in WT and HCN2 cKO (n= 7, 8 mice, respectively). (D) Means of ACh efflux in the first four bins with aCSF and last four bins with neostigmine. Two-way ANOVA RM. Genotype X Treatment F [ , ] = 0.8; P> 0.05. Genotype F [ , ] = 1.0; P> 0.05. Treatment F [ , ] = 11.33; P= 0.005. N.S. not significant vs. WT. (E) Line graph of ACh efflux during cocaine (1 µM) and neostigmine (500 nM) dialysis in WT and cKO (n= 7, 8), plotted as percentage of that during neostigmine. Two-way RM ANOVA. Genotype X Time F [3,39] = 0.65; P> 0.05. Genotype F [ , ] = 13.59; P= 0.002. Time F [ , ] = 1.29; P> 0.05. * p = 0.017 by Bonferroni. (F) Dot plot of means. ** p = 0.003 by unpaired t-test.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Control

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Basal levels of dopamine, DOPAC and HVA in WT and HCN2 cKO mice (n=6, 10 mice, n=6, 7 mice, and n=6, 7 mice, respectively), which were obtained as the average of three stable baseline samples before rewarding stimuli. DA t (10) = 0.1358, p > 0.05; DOPAC, t (7) = 0.7281, p > 0.05; HVA, t (10) = 0.1164, p > 0.05, by Welch’s t-test. (B-D). Responses to exposure to palatable food (B), an unfamiliar male mouse (C), and an unfamiliar female mouse (D) in WT and cKO mice (n = 5, 5 mice, n = 5, 6 mice and n = 5, 5 mice, respectively). The values obtained after rewarding stimuli were compared with the basal levels obtained as the average of three stable baseline samples (100%) using mixed linear models with time as a covariate, and post hoc Bonferroni. * p < 0.05, ** p < 0.01, *** p < 0.001 vs the basal levels. (B) Two Way ANOVA. Group X Time F [7, 64] = 3.04, P= 0.008. Group F [1, 64] = 26.57, P< 0.0001. Time F [7, 64] = 12.13, P< 0.0001. † p = 0.030 †† p = 0.0001 vs WT by Sidak. (C) Two Way ANOVA. Group X Time F [7, 72] = 2.27, P= 0.038. Group F [1, 72] = 1.50, P> 0.05. Time F [7, 72] = 3.59, P= 0.002. † p = 0.033 vs WT by Sidak. (D) Two Way ANOVA. Group X Time F [7, 64] = 3.30, P= 0.005. Group F [1, 64] = 29.48, P< 0.0001. Time F [7, 64] = 6.80, P< 0.0001. † p = 0.026, †† p = 0.001, ††† p < 0.001 vs. WT by Sidak. (E, F) Responses to NAc infusion of cocaine at the concentrations of 1 μM (E) and 10 μM (F) in WT and cKO mice (n= 5, 5 mice and n=5, 5 mice, respectively). (E) Two Way ANOVA. Group X Time F [7, 64] = 0.5866, P> 0.05. Group F [1, 64] = 4.54, P= 0.036. Time F [7, 64] = 10.82, P< 0.0001. (F) Two Way ANOVA. Group X Time interaction F [7, 64] = 0.20, P> 0.05. Group F [1, 64] = 0.61, P> 0.05. Time F [7, 64] = 29.09, P < 0.0001.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques:

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Study design and representative microscope images showing the localization of the fiber trace (arrows) and the dLight1.3 GFP in the NAc. AC, anterior commissure. Scale bars, 500 µm. (B) Representative photometric traces between 5s before and 10s after intraperitoneal injection of saline or cocaine (1.5 mg/kg). Arrows indicate injection times, dashed line represents %ΔF/F= 0. (C) Perievent time heatmap of individual samples(top) and histogram of group means (bottom) of saline or cocaine in n= 3 mice / group. (D) Effect of cocaine (1.5 mg/kg) injections in WT (n= 3 mice) on fluorescent peak amplitude (top) and area (AUC, bottom). * p = 0.018, *** p = 0.0003, vs. saline by unpaired t-test. (E) Study design and representative traces of two consecutive injections of cocaine (1.5 mg/kg) in WT and HCN2 cKO mice. (F) Changes in fluorescent peak amplitude and area by consecutive cocaine injections in WT (n= 8 mice) and cKO (n= 6 mice). Amplitude Two Way ANOVA RM, F genotype X treatment [ , ] = 7.39, P= 0.019. ** p = 0.007 between genotypes by Sidak. Area Two Way ANOVA RM, F genotype X treatment [ , ] = 1.053, P> 0.05. F genotype [ , ] = 6.65, P= 0.024. F treatment [ , ] = 0.53, P> 0.05. * p = 0.026 between genotypes by Sidak. (G) Study design and representative traces following injections of cocaine (1.5 mg/kg) followed by saline or donepezil (DON, 0.2 or 2 mg/kg) in WT and HCN2 cKO. (H) Perievent time heatmap and histogram of averages by saline (n= 3, WT), donepezil 0.2 mg/kg (n= 3, WT) or 2 mg/ kg in WT or cKO (n= 4 mice each). (I) Changes in fluorescent peak amplitude and area in WT. One Way ANOVA (amplitude), P= 0.0002, *** p < 0.001 by Tukey. One Way ANOVA (area), P= 0.003, ** p < 0.01 by Tukey. (J) Effect of donepezil (2 mg/kg) injections in WT and cKO (n= 4, 4 mice) on fluorescent peak amplitude and area. ** p = 0.004, *** p < 0.001 by unpaired t-test.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Microscopy, Injection, Saline

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Distance traveled in WT and cKO during 60 min after cocaine (15 mg/kg, n= 6, 5) or saline (n= 8, 7). Cocaine Two Way ANOVA RM, F genotype X time [5, 45] = 7.68, P< 0.0001, F genotype [ , ] = 14.57, P= 0.004. F time [ , ] = 9.98, P= 0.001. Saline Two Way ANOVA RM, F genotype X time [5, 65] = 4.94, P< 0.0007, F genotype [ , ] = 1.87, P> 0.05. F time [ , ] = 20.92, P< 0.0001. (B) CPP design. Black indicates paradigm phases and their day numbers, and cyan indicates tests names and their day numbers. Acq, acquisition. E, extinction. R, reinstatement. (C) Time spent in each chamber in WT and cKO (n= 8, 5) during the different tests. Two Way ANOVA RM, F genotype X phase [18, 128] = 4.96, P< 0.0001, F genotype [ , ] = 4.75, P= 0.010. F phase [3, 61] = 0.84, P= 0.476. **** p < 0.0001,*** p < 0.001, ** p < 0.01, * p < 0.05 by Tukey. (D) Representative low (left) and high (right) magnification immunohistochemical images following stereotaxic injection of AAV.FLEX.HCN2 in NAc of cKO mouse. Arrows indicate HCN2 labeling in ChI. Scale bars, 500 μm (left) and 50 μm (right). AC, anterior commissure. (E) Time spent in each chamber during CPP acquisition test in cKO mice injected with AAV.FLEX.GFP (n= 9) or AAV.FLEX.HCN2 (n= 11). Two Way ANOVA, F AAV X chamber [ , ] = 5.10, P= 0.037, F AAV [ , ] = 3.73, P> 0.05. F chamber [ , ] = 12.08, P= 0.004. * p = 0.011 by Sidak. (F) Correlation between time in the saline-associated chamber and number of HCN2 immunopositive ChIs in cKO mice injected with AAV.FLEX.HCN2 (n= 6, indicated as full red circles in E). * p = 0.021 by Pearson.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Saline, Immunohistochemical staining, Injection, Labeling

Journal: bioRxiv

Article Title: HCN2 in cholinergic interneurons of the nucleus accumbens mediates reward response

doi: 10.1101/2021.09.21.460269

Figure Lengend Snippet: (A) Representative immunohistochemical images depicting GFP labeling following stereotaxic injection of AAV.FLEX.GFP in the NAc of ChAT Cre mouse. Scale bars, 500 μm. AC, anterior commissure. (B) Time spent in the saline- and cocaine-associated chambers during CPP acquisition (Acq) extinction (E1) and cocaine-induced reinstatement (R1) tests in ChAT Cre mice injected with AAV.FLEX.GFP (n= 8) or AAV.FLEX.HCN2 (n= 11). Two Way ANOVA, F phase X chamber [6, 64] = 5.25, P= 0.0002. F phase [2, 71] = 4.58, P= 0.014. F chamber [3, 36] = 25.41, P< 0.0001. ** p < 0.01 by Tukey. (C-E) Chemogenetic manipulation of NAc ChIs. (C) Representative immunohistochemical images depicting mCherry labeling following stereotaxic injection of AAV2-hSyn-DIO-mCherry to the NAc of ChAT Cre mouse. Scale bars, 500 μm. AC, anterior commissure; LV, lateral ventricle. (D) Study design. CPP conditioning started three weeks after the stereotaxic AAV injection. CNO (4 mg/kg) was injected to all animals on days 7 and 9 of the conditioning phase, 15 min before cocaine. (E) Time spent in the saline- and cocaine-associated chambers during the CPP acquisition test (Acq) in ChAT Cre mice injected with Gi-DREADD, Gs-DREADD or mCherry control (n= 5 mice in each group). Two Way ANOVA, F genotype X chamber [2, 24] = 7.13, P= 0.0037. F genotype [2, 24] = 5.03, P= 0.015. F chamber [1, 24] = 118.20, P< 0.0001. * p = 0.038, ** p = 0.0012 by Tukey.

Article Snippet: Proteins were detected using antibodies for HCN2 (14957) and β-Actin (4970, Cell Signaling Inc., Danvers, MA).

Techniques: Immunohistochemical staining, Labeling, Injection, Saline, Control

Journal:

Article Title:

doi:

Figure Lengend Snippet: Primer sequences for RT‐PCR expression analysis of HCN channels

Article Snippet: Thereafter, tissue was permeabilized with 0.25% Triton X‐100 in PBS for 10 min following by blocking with 2% bovine serum albumin (BSA) in PBS for 20 min. Tissue was incubated overnight in a dark, humidified chamber at 4°C with 1:50 rabbit polyclonal anti‐HCN2 (APC‐030‐AR; Alomone, Israel) preconjugated to ATTO‐594 fluorophore.

Techniques: Expressing, Amplification, Sequencing

Journal:

Article Title:

doi:

Figure Lengend Snippet: Human lymphatic vessels predominantly express HCN2. (a) RT‐PCR analysis of HCN2 (229 base pairs) and HCN3 (589 bp) amplified from thoracic duct (TD) and mesenteric lymphatic vessels (ML); HCN1 (597 bp) and HCN4 (232 bp) were consistently amplified from the control human RNA, dorsal root ganglia (DRG) and heart, respectively, while all lymphatic samples were negative. Samples are presented pairwise as reverse transcriptase positive (+) followed by reverse transcriptase negative (−). HCN2 immunoreactivity (red fluorescence, left panels) was observed in smooth muscle cells of (b) human thoracic duct and (c) mesenteric lymphatic vessels. (d) Antibody specificity was confirmed by the absence of staining when the antibody was preincubated with peptide control (green fluorescent nuclear stain). Scale bars denote 50 μm (top) and 100 μm (middle and bottom), * indicates lumen, and right panels present differential interference contrast (DiC) images of the same section.

Article Snippet: Thereafter, tissue was permeabilized with 0.25% Triton X‐100 in PBS for 10 min following by blocking with 2% bovine serum albumin (BSA) in PBS for 20 min. Tissue was incubated overnight in a dark, humidified chamber at 4°C with 1:50 rabbit polyclonal anti‐HCN2 (APC‐030‐AR; Alomone, Israel) preconjugated to ATTO‐594 fluorophore.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Fluorescence, Staining

Journal:

Article Title:

doi:

Figure Lengend Snippet: HCN transcript expression profile for human thoracic duct (TD) and mesenteric lymphatic vessels (MLV)

Article Snippet: Thereafter, tissue was permeabilized with 0.25% Triton X‐100 in PBS for 10 min following by blocking with 2% bovine serum albumin (BSA) in PBS for 20 min. Tissue was incubated overnight in a dark, humidified chamber at 4°C with 1:50 rabbit polyclonal anti‐HCN2 (APC‐030‐AR; Alomone, Israel) preconjugated to ATTO‐594 fluorophore.

Techniques: Expressing

Journal: Journal of Traditional Chinese Medicine

Article Title: Hyperpolarization-activated cyclic nucleotide-gated 2 contributes to electroacupuncture analgesia on lumbar disc herniation-induced radicular pain through activation of microglia in spinal dorsal horn

doi: 10.19852/j.cnki.jtcm.2022.03.004

Figure Lengend Snippet: Primers used in this study

Article Snippet: After SDS-PAGE electrophoresis and transfer, sections were block with 5% skim milk for 2 h, add primary antibody HCN2 (1∶200, Santa Cruz Biotech., Santa Cruz, CA, USA) and incubate overnight at 4 °C, add followed by goat anti-rabbit secondary antibody (1∶200, Abcam) and incubate for 1 h. The protein bands were visualized on the film with Immobilon Western chemiluminescent HRP substrate (WBKLS0500, MilliporeSigma, Billerica, MA, USA), taken by X-ray photo, β-actin served as an internal reference protein.

Techniques: Sequencing

Journal: Journal of Traditional Chinese Medicine

Article Title: Hyperpolarization-activated cyclic nucleotide-gated 2 contributes to electroacupuncture analgesia on lumbar disc herniation-induced radicular pain through activation of microglia in spinal dorsal horn

doi: 10.19852/j.cnki.jtcm.2022.03.004

Figure Lengend Snippet: EA was given on the 3rd day after modeling, the expression of HCN2 and CX3CL1 was detected on the 6th day. A: the level of HCN2 mRNA in the right L5 dorsal root ganglion; B: the level of CX3CL1 mRNA in the ipsilateral spinal dorsal horn, separately; C, D: HCN2 protein and CX3CL1 protein expression (n = 3). Compared with normal group, aP < 0.05, compared with the acupuncture group, bP < 0.05 (n = 7).

Article Snippet: After SDS-PAGE electrophoresis and transfer, sections were block with 5% skim milk for 2 h, add primary antibody HCN2 (1∶200, Santa Cruz Biotech., Santa Cruz, CA, USA) and incubate overnight at 4 °C, add followed by goat anti-rabbit secondary antibody (1∶200, Abcam) and incubate for 1 h. The protein bands were visualized on the film with Immobilon Western chemiluminescent HRP substrate (WBKLS0500, MilliporeSigma, Billerica, MA, USA), taken by X-ray photo, β-actin served as an internal reference protein.

Techniques: Expressing

Journal: Journal of Traditional Chinese Medicine

Article Title: Hyperpolarization-activated cyclic nucleotide-gated 2 contributes to electroacupuncture analgesia on lumbar disc herniation-induced radicular pain through activation of microglia in spinal dorsal horn

doi: 10.19852/j.cnki.jtcm.2022.03.004

Figure Lengend Snippet: HCN2 antagonist (50 μg of a rat) were injected into subarachnoid space of LDH model rats, per day for 3 d. A: the analgesic effect of EA on mechanical pain was significantly reduced; B: the expression of CX3CL1 in the spinal cord decreased; C, D: Western blot showed that HCN2 protein and CX3CL1 protein expression of the ZD7288 + sham EA group was higher than that of the ZD7288 + EA group, respectively (n = 3). Compared with EA group, aP < 0.05 (n = 7).

Article Snippet: After SDS-PAGE electrophoresis and transfer, sections were block with 5% skim milk for 2 h, add primary antibody HCN2 (1∶200, Santa Cruz Biotech., Santa Cruz, CA, USA) and incubate overnight at 4 °C, add followed by goat anti-rabbit secondary antibody (1∶200, Abcam) and incubate for 1 h. The protein bands were visualized on the film with Immobilon Western chemiluminescent HRP substrate (WBKLS0500, MilliporeSigma, Billerica, MA, USA), taken by X-ray photo, β-actin served as an internal reference protein.

Techniques: Injection, Expressing, Western Blot