Journal: FEBS Open Bio

Article Title: Identification of sodium homeostasis genes in Camelus bactrianus by whole transcriptome sequencing

doi: 10.1002/2211-5463.13380

Figure Lengend Snippet: RNA interference and ROS detection. (A) The experimental formulas of achieved 70% transfection efficiency of shRNA lentiviral vectors targeting SLC6A1 , PCBP2, and PEX5L in renal medulla cells of camel in vitro with lentivirus‐NC/media = 1/5 (200×). Scale bar, 100 μm. (B) Expression levels of SLC6A1 , PCBP2, and PEX5L in the light of the ΔΔCt method in different groups. GAPDH was used as a loading control (intrinsic control), and expression levels were normalized with SLC6A1 , PCBP2, and PEX5L mRNA levels of NC and RNA interference groups. (C) Western blot targeting PCBP2. β‐actin was used as a loading control. (D) ROS detection after shRNA interference in renal medulla cells of camel by red fluorescent imaging DHE probe (100×). Scale bar, 100 μm. (E,F) Fluorescence intensity of ROS after shRNA interference against SLC6A1 , PCBP2, and PEX5L , and after small RNA interference against mixed SLC6A1 , PCBP2, and PEX5L (sRNA‐MIX). Data are presented as mean ± SD ( n = 3) by Student’s t‐test. Symbols: *** P < 0.001. shSLC6A1, shPCBP2, and shPEX5L represent short hairpin RNAs of SLC6A1, PEX5L, and PCBP2 in turn. IC refers to intrinsic control with GAPDH, NC indicates negative control with nonsense shRNA, and BC means blank control.

Article Snippet: SLC6A1, PCBP2, and PEX5L protein expression were severally examined using antibodies against‐SLC6A1 (ab426 and ab72448, Abcam, UK) and PCBP2 (ab96169, Abcam, UK) and PEX5L (nbp1‐91484, Novus Biologicals, USA).

Techniques: Transfection, shRNA, In Vitro, Expressing, Control, Western Blot, Imaging, Fluorescence, Negative Control

Journal: FEBS Open Bio

Article Title: Identification of sodium homeostasis genes in Camelus bactrianus by whole transcriptome sequencing

doi: 10.1002/2211-5463.13380

Figure Lengend Snippet: Salt‐resistance metabolism combined with competing endogenous RNAs (LNC003834, miRNA‐34a, and SLC14A1) and transcribed antioxidant genes ( SLC6A1 , PCBP2, and PEX5L ) in renal medulla of camel.

Article Snippet: SLC6A1, PCBP2, and PEX5L protein expression were severally examined using antibodies against‐SLC6A1 (ab426 and ab72448, Abcam, UK) and PCBP2 (ab96169, Abcam, UK) and PEX5L (nbp1‐91484, Novus Biologicals, USA).

Techniques:

Journal: eLife

Article Title: Alterations in the intrinsic properties of striatal cholinergic interneurons after dopamine lesion and chronic L-DOPA

doi: 10.7554/eLife.56920

Figure Lengend Snippet: ( A ) Representative perforated-patch recordings of sAP from sham, 6-OHDA and +chronic-LD groups. Dotted line represents a threshold potential, which was similar between the groups. Note the markedly slower rate of cell depolarization after the action potential in the ChIs from 6-OHDA group and decreased amplitude of the AHP in a neuron from the +chronic-LD group. ( B ) Representative cell-attached recordings of ChI activity before and after treatment with HCN channel blocker ZD7288 (25 µM). ( C ) Current-clamp protocol (lower) and representative recordings (upper) showing voltage sag, a characteristic of HCN channel activation. ( D ) Quantification of sag amplitude at different current steps. Sham n = 39 neurons/18 mice, 6-OHDA n = 23 neurons/11 mice, chronic-LD n = 30 neurons/12 mice; p<0.05 (*) for 6-OHDA vs. two other groups, p<0.05 (#) 6-OHDA vs. chronic-LD at indicated current by Tukey’s multiple comparisons test following repeated measures two-way ANOVA. ( E ) Voltage-clamp protocol (lower) and representative ZD7288-sensitive ( I h ) current (upper). ( F ) I h density was decreased in ChIs from DA-depleted mice. Sham n = 11 neurons/6 mice, 6-OHDA n = 12 neurons/6 mice, chronic-LD n = 13 neurons/7 mice; p<0.05 (*) for sham vs. 6-OHDA at −100 mV by Tukey’s multiple comparison following repeated measures two-way ANOVA. ( G ) Boltzmann fits of normalized I h densities. (Same N as in F); p<0.05 (*) for V50 values of sham vs. 6-OHDA by Tukey’s multiple comparison following one-way ANOVA. ( H ) ChI-specific gene expression of Hcn1-4 isoforms and Pex5l (Trip8b) measured by RT-qPCR from striatal mRNA immunoprecipitated from Chat-Cre:Rpl22 HA (ribotag) mice treated as indicated. Target mRNA levels were normalized to β-actin. Sham n = 6 samples/20 mice, 6-OHDA n = 4 samples/17 mice, chronic-LD n = 6 samples/22 mice; P-values on graphs are for Kruskal-Wallis test, p<0.05 (*) with Dunn’s multiple comparisons test. Figure 3—source data 1. Individual neuron data and statistics for all panels.

Article Snippet: The probes were obtained from Applied Biosystems and include: Hcn1 (Mm00468832_m1), Hcn2 (Mm00468538_m1), Hcn3 (Mm01212852_m1), Hcn4 (Mm01176086_m1), Pex5l (Mm00458088_m1), Chat (Mm01221882_m1), Kcnn1 (Mm01349167_m1), Kcnn2 (Mm00446514_m1), Kcnn3 (Mm00446516_m1) and Actb (4352341E).

Techniques: Activity Assay, Activation Assay, Comparison, Gene Expression, Quantitative RT-PCR, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b-deficient mice have altered atrial electrophysiology. ( A ) In vitro electrophysiological measurements from Langendorff-perfused hearts show an increase of atrial refractory period (ARP) and atrioventricular-nodal refractory period (AVNRP) in TRIP8b-deficient mice, without changes in sino-nodal activity (sino-nodal recovery time, SNRT) and heart rate (HR). ( B ) Representative tracings are shown for wild-type and TRIP8b-deficient mice. A, atrial activity; atrium electrophysiological tracings from the atrium; V, ventricular activity; ventricle electrophysiological tracings from the ventricle; black arrowheads mark atrial or ventricular stimulation. ( C ) Ganglionic blockade with 0.5 mM hexamethonium leads to a reduction of AVNRP in TRIP8b-deficient mice. Data are presented as box plots (minimum to maximum, n = 5–11 per genotype) and were compared using an unpaired t -test or Mann–Whitney, as appropriate.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: In Vitro, Activity Assay, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNA is present in the cardiac nervous system. ( A ) Exon 6–7 can be amplified in cDNA of ganglia-containing atrial tissue of wild-type but not of TRIP8b-deficient mice (left panel). Quantitative PCR analyses show that exon 8–9, 9–10, and 13–14 of Trip8b are still detectable in knockout mice. Data (normalized to Cdkn1b) are presented as individual data points with SEM ( n = 3, right panel) and were compared via one-way ANOVA followed by Sidaks’ multiple comparison test; ns, not significant. ( B ) Trip8b mRNA can be visualized with RNAscope in situ hybridization in neuronal cell bodies of cardiac ganglia. Black arrows in magnifications point to single neurons with Trip8b mRNAs. ( C ) Trip8b mRNA (black arrows) can be visualized with RNAscope in situ hybridization in cardiac nerves of wild-type mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Amplification, Real-time Polymerase Chain Reaction, Knock-Out, In Situ Hybridization

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: Trip8b mRNAs are detectable in the cardiac conduction system to a lower amount than in the intracardiac nervous system. ( A ) Sinus node and ( B ) atrioventricular node (AV node) were identified via hematoxylin and eosin (H&E) staining and Hcn4 RNAscope in situ hybridization. Subsequent sections treated with a probe specific for Trip8b show solitary mRNA spots (black arrows) surrounding the sinus node artery and in the AV node. Nuclei are counterstained with hematoxylin in blue. ( C ) The histogram shows the distribution of Trip8b mRNA spots per cell in the intracardiac nervous system (ICNS, nerves, and ganglia), sinus node, and AV node of wild-type and TRIP8b-deficient mice. Overall, 279–404 cells were analyzed for each region of interest per genotype, n = 2–3 images/genotype. ( D ) Trip8b in situ hybridization (red) detects mRNA in wild-type mice but also, to a lower amount, in knockout mice.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining, In Situ Hybridization, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the atrial lysates and the cardiac autonomic nervous system. ( A ) Western blot analysis of brain tissue as positive control detects specific bands for TRIP8b (NBP2-38840, Novusbio) already at 2.5 µg total protein. For the heart, 50 µg atrial or ventricular lysate did not show any specific bands, while HCN4 ( B ) is detectable in both genotypes. ( C ) Immunohistochemistry for TRIP8b (APR-070, Alomone Labs) on paraffin sections was established in the central nervous system, more specifically, the cerebral cortex. Neurons positive for TRIP8b are detectable in the wild-type animals but not cortex of TRIP8b-deficient animals. ( D ) To increase the sensitivity of detection, atrial whole-mount preparations (upper panel shows exemplary staining with αTH ab152, Merck Millipore) were stained and ganglia cut out for confocal microscopy (bottom panel with αTH ab76442, Abcam). No specific signal was obtained for TRIP8b (APR-070, Alomone Labs), and no differences were detectable between the genotypes.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Western Blot, Positive Control, Immunohistochemistry, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: TRIP8b protein is not detectable in the cardiac conduction system in wild-type mice. Sinus node (upper panel) and atrioventricular node (AV node, bottom panel) were identified by anatomical landmarks and HCN4 staining (green). No staining for TRIP8b (red, APR-070, Alomone Labs) was detectable beyond the background.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterization of the HCN Interaction Partner TRIP8b/PEX5R in the Intracardiac Nervous System of TRIP8b-Deficient and Wild-Type Mice

doi: 10.3390/ijms22094772

Figure Lengend Snippet: HCN channel expression in intracardiac ganglia. ( A ) In situ hybridization of two exemplary wild-type ganglia for Hcn2 (green) and Hcn4 (red). Both mRNAs are present within the ganglia. Boxed area is magnified in the inlay. ( B ) Gene expression analysis of Hcn2 and Hcn4 in TRIP8b-deficient mice and wild-type littermates. Data are presented as normalized gene expression to Cdkn1b using the formula 2 −ΔCt (box plots, minimum to maximum, n = 6 per genotype) and were compared using Mann–Whitney test.

Article Snippet: This was verified using a different TRIP8b antibody (Alomone Labs, Jerusalem, Israel) with the same result.

Techniques: Expressing, In Situ Hybridization, MANN-WHITNEY

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Illustration depicting the acute corticosterone treatment in the dorsal hippocampus. B Representative dorsal hippocampal slices immunolabeled with antibody against TRIP8b. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic TRIP8b protein expression. C Quantification of TRIP8b protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. D Representative dorsal hippocampal slices immunolabeled with antibody against HCN1. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 protein expression. E Quantification of HCN1 protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. F , G We performed cell-attached voltage-clamp recordings. F Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). G I h was significantly elevated in the dorsal CA1 neurons from corticosterone treatment compared with those from the vehicle-treated group. Dexamethasone reduced R in at RMP ( H ) and at −65 mV ( J ) in dorsal CA1 neurons. Corticosterone-induced decrease in R in at RMP ( H ) and at −65 mV ( J ) was blocked by RU 486, KT5720, and ZD7288. Changes in R in at RMP ( I ) and at −65 mV ( K ) were blocked by RU 486, KT5720, and ZD7288. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Immunolabeling, Expressing

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Timeline of CSDS, behavioral tests, electrophysiology, and biochemical assay. B CSDS produced the susceptible and resilient phenotype during the social interaction test. After 1 month ( C ) or 3 months ( D ) of no CSDS, susceptible mice showed persistent social avoidance during the social interaction test. E , F We performed whole-cell current-clamp recordings. E Representative voltage responses with depolarizing current step (210 pA; 750 ms) at RMP in dorsal CA1 neurons. F Dorsal CA1 neurons of susceptible group had lower action potential firing than control group, whereas the resilient group had higher action potential firing. G , H We performed cell-attached voltage-clamp recordings. G Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). H I h was significantly elevated in the dorsal CA1 neurons from susceptible group compared with those from the control and resilient mice. Representative dorsal hippocampal slices immunolabeled with antibody against HCN1 ( I ) and TRIP8b ( K ). Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 and TRIP8b protein expression. Quantification of HCN1 ( J ) and TRIP8b ( L ) protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. M , N We performed whole-cell current-clamp recordings. M Corticosterone reduced R in at RMP of the dorsal CA1 neurons in the control and resilient groups, but had no effect on R in in susceptible group. N Changes in R in at RMP were much lower in susceptible group compared to the control and resilient groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Produced, Control, Immunolabeling, Expressing