antibodies against unc80  (Bioss)

Journal: International Journal of Molecular Sciences

Article Title: Imbalance in Unc80 RNA Editing Disrupts Dynamic Neuronal Activity and Olfactory Perception

doi: 10.3390/ijms25115985

Figure Lengend Snippet: Expression patterns of ADAR2-mediated RNA editing of Unc80 in the brain and its structural implications. Total RNA was extracted from various tissues and brain regions from wild-type (WT) and Adar2-knockout (KO) mice. The samples were then analyzed using RT-PCR ( A ) and Sanger sequencing ( B ). The sequencing chromatograms highlight the absence of guanine (“G”) signals (indicated by arrows) in the KO samples, demonstrating the reliance of Unc80 editing on ADAR2’s enzymatic function. Differences in RNA editing levels across brain regions were also observed. The percentage represents the editing frequency, calculated by taking the peak area of G peak over the sum of A and G peaks. ( C ) Prediction of 3-dimensional protein structure models of Unc80 WT and Unc80 S2732G . The magnified view of the region of interest highlights the residue change from Ser to Gly due to editing. ( D ) Immunofluorescence analysis on olfactory bulb coronal sections, specifically localizing Unc80 (green) and NeuN (red) protein. The a and b correspond to magnified views of the white dashed boxes in left panel. The arrows indicate the positions of overlapping fluorescence (green and red). Scale bars = 200 µm in the left panel and 40 µm in the magnified images. (TA: Tibialis anterior muscle, GA: Gastrocnemius muscle, CT: cortex, CB: cerebellum, HP: hippocampus, OB: olfactory bulb).

Article Snippet: Sagittal frozen sections (30 μm) were cut with a cryostat (Leica Microsystems, Wetzlar, Germany, SM 2010R), permeabilized, and blocked with 10% BSA in 0.5% Triton X-100 for 2 h, then incubated overnight at 4 °C with primary antibodies against Unc80 (BS-12121R, BIOSS, Woburn, MA, USA) and NeuN (MAB377, Merck Millipore, Darmstadt, Germany).

Techniques: Expressing, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Sequencing, Residue, Immunofluorescence, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Imbalance in Unc80 RNA Editing Disrupts Dynamic Neuronal Activity and Olfactory Perception

doi: 10.3390/ijms25115985

Figure Lengend Snippet: Generation of Unc80 -deleted and site-specific RNA editing mouse models: ( A ) Experimental schematic of the CRISPR/Cas9-based genetic engineering to generate deficient and knock-in mouse models. gRNA (nucleotide with orange background) together with Cas9 created indels, thus establishing knockout mice. In parallel, the addition of synthesized homologous DNA templates corresponding to the Unc80 sequence region with a substitution for the “pre-edited” (G-form) allele (gain of editing) or the “unedited” version (loss of editing) resulted in the knock-in models. ( B ) Breeding schemes of mice with site-specific knock-ins at the Unc80 editing site. ( C ) Genomic DNA sequencing for genotyping demonstrates the WT sequence (top) and mutations in founder strains ( Unc80 +/− ). Deletions and point mutations are indicated by a red background and purple boxes, respectively, in heterozygous deletion (Unc80 +/− ) or knock-in mice ( Unc80 S/S , Unc80 G/G , and Unc80 S/G ). The green box denotes the targeted editing site. ( D ) Gross morphological comparison and body weights of newborn (P0) mice across genotypes. ( E ) Offspring genotyping from heterozygous Unc80 +/− crosses reveals a lower-than-expected birth rate for homozygous deletion offspring, whereas knock-in alleles followed an approximately Mendelian inheritance pattern. ( F,G ) Unc80 protein and mRNA levels in brain tissue lysates (cortex and olfactory bulb) from CRISPR-engineered Unc80 -deficient mice were assessed via immunoblotting ( F ) and qRT-PCR (OB, ( G )).

Article Snippet: Sagittal frozen sections (30 μm) were cut with a cryostat (Leica Microsystems, Wetzlar, Germany, SM 2010R), permeabilized, and blocked with 10% BSA in 0.5% Triton X-100 for 2 h, then incubated overnight at 4 °C with primary antibodies against Unc80 (BS-12121R, BIOSS, Woburn, MA, USA) and NeuN (MAB377, Merck Millipore, Darmstadt, Germany).

Techniques: CRISPR, Knock-In, Knock-Out, Synthesized, Sequencing, DNA Sequencing, Comparison, Western Blot, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Imbalance in Unc80 RNA Editing Disrupts Dynamic Neuronal Activity and Olfactory Perception

doi: 10.3390/ijms25115985

Figure Lengend Snippet: MEMRI-based neuronal activity assessment in the olfactory systems of Unc80 S/S and Unc80 G/G mice: ( A ) Anatomical MRI images and corresponding color mapping generated during odor stimulation. ( B ) Variation in MEMRI signal intensity in response to odor stimulation, with error bars representing the SD. ( C ) In vivo glutamate- and dopamine-sensitive CEST-MRI images showing anatomical and color mapping in various brain nuclei. ( D ) Quantitative changes in CEST-MRI signals across different brain nuclei for Unc80 S/S and Unc80 G/G mice, depicting both glutamate (left) and dopamine (right) contrasts. Brain regions assessed include the olfactory bulb (OB), anterior olfactory nucleus (AON), and anterior piriform cortex (APC). Error bars indicate SD. Statistical significance is denoted as follows: ns (not significant); p > 0.05; * p < 0.05; ** p < 0.01.

Article Snippet: Sagittal frozen sections (30 μm) were cut with a cryostat (Leica Microsystems, Wetzlar, Germany, SM 2010R), permeabilized, and blocked with 10% BSA in 0.5% Triton X-100 for 2 h, then incubated overnight at 4 °C with primary antibodies against Unc80 (BS-12121R, BIOSS, Woburn, MA, USA) and NeuN (MAB377, Merck Millipore, Darmstadt, Germany).

Techniques: Activity Assay, Generated, In Vivo

Journal: International Journal of Molecular Sciences

Article Title: Imbalance in Unc80 RNA Editing Disrupts Dynamic Neuronal Activity and Olfactory Perception

doi: 10.3390/ijms25115985

Figure Lengend Snippet: Neurophysiological connection of Unc80 editing event to olfactory sensing and motor control: Habituation and dishabituation behaviors, in response to odors, were analyzed for Unc80 knockout and site-specific editing variants ( A , B ), along with odor-induced neuronal activity assays ( C , D ). Mice with different genotypes (wild-type vs. Unc80 +/− in ( A ), Unc80 S/S vs. Unc80 G/G in ( B )) were exposed to odors, and their explorative times near the odor source were recorded and presented as mean ± SD. The cohorts consisted of: WT (n = 13), Unc80 +/− (n = 5), Unc80 S/S (n = 9), Unc80 G/G (n = 8). For the neuronal activation assay, knock-in mice with site-specific edits were either not exposed or exposed to banana oil for up to 60 min and subsequently sacrificed for olfactory bulb isolation, from which total RNA and proteins were prepared for qRT-PCR ( C ) and Western blot ( D ) analyses, respectively. Changes in the expression of c-Fos were monitored as a readout for neuronal activation. The bar graph represents the relative mRNA expression levels of Fos. Statistical significance is indicated as follows: ns (not significant); p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Sagittal frozen sections (30 μm) were cut with a cryostat (Leica Microsystems, Wetzlar, Germany, SM 2010R), permeabilized, and blocked with 10% BSA in 0.5% Triton X-100 for 2 h, then incubated overnight at 4 °C with primary antibodies against Unc80 (BS-12121R, BIOSS, Woburn, MA, USA) and NeuN (MAB377, Merck Millipore, Darmstadt, Germany).

Techniques: Control, Knock-Out, Activity Assay, Activation Assay, Knock-In, Isolation, Quantitative RT-PCR, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Imbalance in Unc80 RNA Editing Disrupts Dynamic Neuronal Activity and Olfactory Perception

doi: 10.3390/ijms25115985

Figure Lengend Snippet: Transcriptomic analysis of the olfactory bulb in Unc80 editing variant mice. Transcriptome-wide RNA-seq was conducted to identify changes in the olfactory bulb of Unc80 S/S and Unc80 G/G mice. ( A – C ) The overall transcriptome distribution is represented in a principal component analysis (PCA) plot ( A ) and a volcano plot ( B ), delineating genotype-specific gene expression profiles. A heatmap ( C ) displays genes with significant differential expression (|fold-change| > 1.5, p < 0.05; n = 6 per genotype, with samples pooled from three mice each) between the two strains. ( D ) Bubble chart of the enriched canonical pathway from IPA analysis. The x -axis represents the significance ( p -value), and the y -axis shows the top 17 significantly enriched pathways based on differentially expressed genes (DEGs). The size of the circles corresponds to the number of genes associated with each pathway. The colors of the bubbles denote the Z-score, indicating whether the pathway is activated (orange, positive Z-score) or inhibited (blue, negative Z-score). ( E ) Normalized read count plots highlight expression variations in gene sets linked to neuronal signaling between Unc80 S/S and Unc80 G/G mice. Statistical significance in this figure is indicated as follows: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Sagittal frozen sections (30 μm) were cut with a cryostat (Leica Microsystems, Wetzlar, Germany, SM 2010R), permeabilized, and blocked with 10% BSA in 0.5% Triton X-100 for 2 h, then incubated overnight at 4 °C with primary antibodies against Unc80 (BS-12121R, BIOSS, Woburn, MA, USA) and NeuN (MAB377, Merck Millipore, Darmstadt, Germany).

Techniques: Variant Assay, RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a Illustrator and workflow for single-cell RNA sequencing (scRNA-seq) of SAN. b t -distributed stochastic neighbor embedding (t-SNE) analysis identified five clusters including four SAN cell clusters (Clusters 1, 2, 3, 4) and one atrial and ventricular (AV) cardiomyocytes cell cluster (Cluster 5) ( n = 771 biologically independent cells of 25 independent animals). c Heat map shows top differentially expressed genes (DEGs) of each cluster. Cluster 4 was highly expressed cell markers including Hcn4, Hcn1 , and Ednrb, Pde1a and identified as the core cell cluster. d Feature plot of DEGs shows Vsnl1, Unc80 , and Dlgap1 were expressed more specifically than Hcn4 and Hcn1 in Cluster 4. Location of each cluster was marked with the red circle. e Gene Ontology (GO) terms show the biological processes of SAN cell clusters and Cluster 4 was mainly associated with regulation of heart rate and ion transmembrane transport, while Cluster 1, 2, and 3 were all related to electrical activity but had certain difference.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: RNA Sequencing Assay, Activity Assay

Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a qPCR analysis shows the expression of Hcn4, Vsnl1, Dlgap1, and Unc80 in sinoatrial node (SAN), atrial, and ventricular tissue, respectively ( n = 4 biologically independent animals per group), Dunnett’s multiple comparisons test, data are represented as mean ± s.e.m., adjusted p value was labeled on the top. RA right atrium, LA left atrium, RV right ventricle, LV left ventricle. b – g Immunohistochemical analysis of VSNL1, DLGAP1, and UNC80 in SAN slices at low magnification. Representative images are shown from n = 3 biologically independent samples. b Immunofluorescence images of HCN4 (green), VSNL1 (red), and DAPI (blue). c Immunofluorescence images of HCN4 (green), DLGAP1 (red), and DAPI (blue). d Immunofluorescence images of HCN4 (green), UNC80 (red), and DAPI (blue). b – d The arrows indicate the tissues surrounding the SAN. Scale bar = 250 μm. e – g Localization of the VSNL1, DLGAP1, and UNC80 within SAN at high magnification. e Immunofluorescence images of HCN4 (green), VSNL1 (red), and DAPI (blue). f Immunofluorescence images of HCN4 (green), DLGAP1 (red), and DAPI (blue). g Immunofluorescence images of HCN4 (green), UNC80 (red), and DAPI (blue). Scale bar = 50 μm. e – g Zoom images (the red box regions in the merged image) show the higher magnification. Scale bar = 10 μm.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: Expressing, Labeling, Immunohistochemical staining, Immunofluorescence

Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a Illustrator and workflow for single-cell RNA sequencing (scRNA-seq) of SAN. b t -distributed stochastic neighbor embedding (t-SNE) analysis identified five clusters including four SAN cell clusters (Clusters 1, 2, 3, 4) and one atrial and ventricular (AV) cardiomyocytes cell cluster (Cluster 5) ( n = 771 biologically independent cells of 25 independent animals). c Heat map shows top differentially expressed genes (DEGs) of each cluster. Cluster 4 was highly expressed cell markers including Hcn4, Hcn1 , and Ednrb, Pde1a and identified as the core cell cluster. d Feature plot of DEGs shows Vsnl1, Unc80 , and Dlgap1 were expressed more specifically than Hcn4 and Hcn1 in Cluster 4. Location of each cluster was marked with the red circle. e Gene Ontology (GO) terms show the biological processes of SAN cell clusters and Cluster 4 was mainly associated with regulation of heart rate and ion transmembrane transport, while Cluster 1, 2, and 3 were all related to electrical activity but had certain difference.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: RNA Sequencing Assay, Activity Assay

Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a qPCR analysis shows the expression of Hcn4, Vsnl1, Dlgap1, and Unc80 in sinoatrial node (SAN), atrial, and ventricular tissue, respectively ( n = 4 biologically independent animals per group), Dunnett’s multiple comparisons test, data are represented as mean ± s.e.m., adjusted p value was labeled on the top. RA right atrium, LA left atrium, RV right ventricle, LV left ventricle. b – g Immunohistochemical analysis of VSNL1, DLGAP1, and UNC80 in SAN slices at low magnification. Representative images are shown from n = 3 biologically independent samples. b Immunofluorescence images of HCN4 (green), VSNL1 (red), and DAPI (blue). c Immunofluorescence images of HCN4 (green), DLGAP1 (red), and DAPI (blue). d Immunofluorescence images of HCN4 (green), UNC80 (red), and DAPI (blue). b – d The arrows indicate the tissues surrounding the SAN. Scale bar = 250 μm. e – g Localization of the VSNL1, DLGAP1, and UNC80 within SAN at high magnification. e Immunofluorescence images of HCN4 (green), VSNL1 (red), and DAPI (blue). f Immunofluorescence images of HCN4 (green), DLGAP1 (red), and DAPI (blue). g Immunofluorescence images of HCN4 (green), UNC80 (red), and DAPI (blue). Scale bar = 50 μm. e – g Zoom images (the red box regions in the merged image) show the higher magnification. Scale bar = 10 μm.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: Expressing, Labeling, Immunohistochemical staining, Immunofluorescence