anti trpv1 vr1 polyclonal antibody rabbit  (Alomone Labs)

Journal: bioRxiv

Article Title: GABA-induced Ca 2+ signaling in the primary cilium of neurons

doi: 10.1101/2025.05.26.656109

Figure Lengend Snippet: A. Immunofluorescence images of mouse hippocampal CA1 region showing the distribution of Arl13b (magenta, primary cilium), AC3 (green, neuronal primary cilium) and NeuN (neuronal marker). Arrows show the location of non-neuronal primary cilia. (scale bar 10 µm). B. Quantification of cilia length in neuronal and non-neuronal cells in the mouse CA1 (13,38± 0,36µm n=40 neuron and 8,26± 0,40µm n=14 glia, two tailed t-test P<0,0001). C. Immunofluorescence images of mouse CA1 showing the distribution of GABA-B1 receptors (magenta), AC3 (green; left column), Arl13b (green; right column) and NeuN (blue; neurons) or GFAP (blue; glial cells). D. Magnifications of boxed region in C showing the presence of GABA-B1 receptors on primary cilia in neurons but not in glial cells. Fluorescence intensity along the dashed lines is shown in E below. E. Line profiles drawn along a cilium from a neuron (left) and a glial cell (right) showing the presence of GABA-B1 receptors at the cilia base of neuronal primary cilia. F. STED microscopy image of a neuronal cilia showing the presence of GABA-B1 receptors at the cilia base, where it colocalized with the cilia membrane marker AC3. The transverse profiles shown below represents the fluorescence intensity along the dashed line. G. Immunofluorescence image showing the localization of GABA-B1 receptors to the primary cilium base (arrowheads) and to dendrites (arrows). H. GABA-B1 receptor enrichment along the cilia base and a corresponding segment from an adjacent dendrite (n=10). I. Relative GABA-B1 receptor enrichment in the primary cilium and dendrites of neurons from the mouse CA1 (n=10, P=0,3930 two tailed t-test).

Article Snippet: The following primary antibodies (1/300 dilutions) were used: ARL13b (Abcam ab136648), AC3 (Abcam ab277619), AC3 (Alomone AAR-043), NeuN (Sigma-Aldrich ABN90), GFAP (Synaptic System 173–004), Synapsin II (Alomone ANR-015), GABA-B1 (Alomone AGB-001), MCHR1 (Thermo Fischer PA5-77492), Patched (Abcam ab53715), EP4 (Santa Cruz Biotechnology sc-55596), ANKS6 (Sigma HPA008355), NPHP3 (Proteintech 22026-1-AP), NEK8 (kind gift from Prof. David R. Beier, Seattle Children’s Hospital, USA).

Techniques: Immunofluorescence, Marker, Two Tailed Test, Fluorescence, Microscopy, Membrane

Journal: bioRxiv

Article Title: GABA-induced Ca 2+ signaling in the primary cilium of neurons

doi: 10.1101/2025.05.26.656109

Figure Lengend Snippet: A. Immunofluorescence images from mouse cortex (top) and Striatum (bottom) showing the distribution of GABA-B1 receptors (magenta), Arl13b (green) and neurons (blue). B, C. Quantifications of GABA-B1 immunofluorescence intensity at the cilia base in the cortex, striatum and hippocampus (CA1) in relation to dendrites in the same region (n cilia/dendrites : striatum 7/6, cortex 6/7, CA1 17/16). * P=0,0346, *** P=0,0014; Kruskal-Wallis ANOVA followed by multiple comparisons. D. Length of primary cilia (green) and the GABA-B1 receptor compartment (magenta) in primary cilia of neurons in the cortex, striatum and hippocampus (striatum GABA-B1/ARL13b n=28, cortex GABA-B1/ARL13b n=64, CA1 GABA-B1/ARL13b n=20). *P=0,0189, ***P=0,0001; Kruskal-Wallis ANOVA followed by multiple comparisons. E. Immunofluorescence images from mouse olfactory epithelium showing the distribution of GABA-B1 receptors (magenta), OMP (green; left), CNGA2 (green, right) and nuclei (blue). F. Immunofluorescence images from mouse respiratory epithelium showing the distribution of GABA-B1 receptors (magenta) and acetylated tubulin (motile cilia; green).

Article Snippet: The following primary antibodies (1/300 dilutions) were used: ARL13b (Abcam ab136648), AC3 (Abcam ab277619), AC3 (Alomone AAR-043), NeuN (Sigma-Aldrich ABN90), GFAP (Synaptic System 173–004), Synapsin II (Alomone ANR-015), GABA-B1 (Alomone AGB-001), MCHR1 (Thermo Fischer PA5-77492), Patched (Abcam ab53715), EP4 (Santa Cruz Biotechnology sc-55596), ANKS6 (Sigma HPA008355), NPHP3 (Proteintech 22026-1-AP), NEK8 (kind gift from Prof. David R. Beier, Seattle Children’s Hospital, USA).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: GABA-induced Ca 2+ signaling in the primary cilium of neurons

doi: 10.1101/2025.05.26.656109

Figure Lengend Snippet: A. Immunofluorescence images from mouse hippocampus showing the distribution of MCHR1 (green), AC3 (red) and neurons (yellow). B. Immunofluorescence images from mouse hippocampus showing the distribution of MCHR1, Ptch1 and EP4 in neuronal primary cilia. Quantification of MCHR1, Ptch1 and EP4 enrichment in neuronal primary cilia relative to the local background in the mouse hippocampus, cortex and striatum are shown below. C. Normalized fluorescence intensity profiles along primary cilia of hippocampal neurons showing the distribution (from base to tip) of Arl13b (black), EP4 (red), Ptch (orange), MCHR1 (green) and GABA-B1 (blue). The lines are averages from 13 (EP4), 10 (Ptch), 10 (MCHR1) and 11 (GABA-B1) cilia. D. Cartoon showing the proposed localization of the inversin compartment, composed of ANKS6, INVS, NEK8 and NPHP3, distal to the ciliary transition zone. E. Distribution of NEK8, ANKS6 and NPHP3 in primary cilia of neurons (top), mouse islet β-cells and clonal MIN6 β-cells.

Article Snippet: The following primary antibodies (1/300 dilutions) were used: ARL13b (Abcam ab136648), AC3 (Abcam ab277619), AC3 (Alomone AAR-043), NeuN (Sigma-Aldrich ABN90), GFAP (Synaptic System 173–004), Synapsin II (Alomone ANR-015), GABA-B1 (Alomone AGB-001), MCHR1 (Thermo Fischer PA5-77492), Patched (Abcam ab53715), EP4 (Santa Cruz Biotechnology sc-55596), ANKS6 (Sigma HPA008355), NPHP3 (Proteintech 22026-1-AP), NEK8 (kind gift from Prof. David R. Beier, Seattle Children’s Hospital, USA).

Techniques: Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: GABA-induced Ca 2+ signaling in the primary cilium of neurons

doi: 10.1101/2025.05.26.656109

Figure Lengend Snippet: A. Immunofluorescence images from naïve or 5HT6-GGECO1-expressing cultured mouse neurons (from cortex and CA1) showing the distribution of GABA-B1 receptors (magenta) in primary cilia positive for AC3 (green). Intensity profiles from lines drawn along the cilia are shown to the right and further show the confinement of GABA-B1 receptors to the cilia base of cultured neurons. B. Quantification of GABA-B1 receptor enrichment at the cilia base in naïve cultured neurons or cultured cortical and hippocampal neurons expressing 5HT6-GGECO1 (naïve 2,76±0,37; Cx 8,18±0,91; Hipp 4,94±0,44 arbitrary units). GABA-B1 positive segment length was measured for each group and found significantly different (naïve, Cx and Hipp, in µm: 2,78±0,37; 8,18±0,91; 5,03±0,39; mean±SEM; Kruskal-Wallis ANOVA followed by multiple comparisons, naïve n=12, Cx n=22, Hipp n=38, for A & B). C. Simultaneous recording of cytoplasmic (grey) and ciliary (pink) calcium activities from a neuron before and after stimulation with 100nM baclofen. Activation of metabotropic GABA receptors was without effect on the frequency of somatic events while ciliary signaling was increased by the agonist. Positive deviations from baseline were computed over time to produce ciliary activity count traces (black, lower panel). D. Averaged activity counts corresponding to neurons in control (blue) baclofen (100nM, black) and TTX (1 µM) + baclofen (100nM, grey) showing an increase of activity after stimulation with baclofen. E. Quantification of activity changes was performed in a pair wise fashion, comparing the averaged activity during initial vs final period (control) and baseline vs baclofen (either in the absence of presence of TTX). Time lacked an effect on activity (control P=0,4332) while baclofen stimulation led to a significant increase in both - /+ TTX conditions (respectively P=0,0002 and P=0,0039; two tailed Wilcoxon matched-pairs signed rank test) F. To test whether abolition of action potentials had an impact on spontaneous ciliary activity, recordings in control and TTX (initial 20 minutes both groups) were compared. Statistical analysis showed no difference (P=0,1468 Mann-Whitney two tailed t-test). G. Immunofluorescence images showing the distribution of primary cilia (AC3; green) and synapses (Synapsin II; magenta) in cultured cortical neurons. Boxed areas are magnified to the right. Scatter plot to the left shows Synapsin II fluorescence intensity at primary cilia and random locations within the same sample (P=0,3696 Wilcoxon matched pairs signed rank test).

Article Snippet: The following primary antibodies (1/300 dilutions) were used: ARL13b (Abcam ab136648), AC3 (Abcam ab277619), AC3 (Alomone AAR-043), NeuN (Sigma-Aldrich ABN90), GFAP (Synaptic System 173–004), Synapsin II (Alomone ANR-015), GABA-B1 (Alomone AGB-001), MCHR1 (Thermo Fischer PA5-77492), Patched (Abcam ab53715), EP4 (Santa Cruz Biotechnology sc-55596), ANKS6 (Sigma HPA008355), NPHP3 (Proteintech 22026-1-AP), NEK8 (kind gift from Prof. David R. Beier, Seattle Children’s Hospital, USA).

Techniques: Immunofluorescence, Expressing, Cell Culture, Activation Assay, Activity Assay, Control, Two Tailed Test, MANN-WHITNEY, Fluorescence

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: ( A ) TIRF microscopy images depicting a MIN6 cell expressing EpacS H187 , targeted to the cilium by Smoothened. Images on top are from resting conditions, and images at the bottom are in the presence of 10 µM forskolin. ( B ) Representative recording of EpacSH187 FRET ratio (CFP/YFP) changes in the cilium and cytosol in response to 10 µM forskolin. Below are shown the ratio (means ± SEM) between resting ciliary and cytosolic CFP/YFP FRET ratio in Smo-EpacS H187 ( n = 14 cells from four experiments), 5HT 6 -EpacS H187 ( n = 24 cells from eight experiments) and mArl13b-EpacS H188 ( n = 8 cells in two experiments) expressing MIN6 cells and mArl13b-EpacS H188 ( n = 39 cells in five independent experiments) expressing mouse islet cells. ( C ) Pseudo-colored TIRF images of an islet cell showing an increase in ciliary and cytosolic cAMP in response to 10 µM forskolin and 100 µM IBMX reported with mArl13b-RFlincA. Fluorescence changes over time are shown below. ( D ) TIRF microscopy recordings of Smo-EpacS H187 FRET ratio in the cilium and cytosol of MIN6 cells following addition and washout of forskolin. Data presented as means for ten cells. ( E ) Amplitude-normalized traces from ( D ) highlighting the kinetics during forskolin-evoked cAMP increases in the cilia and cytosol Shown to the right are calculations of the time-constant for the forskolin-induced rise of cAMP. ( F ) Amplitude-normalized traces from ( D ) highlighting the kinetics during cAMP lowering in the cilia and cytosol following forskolin washout. Shown to the right are calculations of the time-constant for the forskolin-induced rise of cAMP. Statistical significance was assessed with the two-tailed paired Student´s t test (no difference). ( G ) Representative confocal microscopy images of mouse islets immunostained against the GLP-1 receptor (yellow), insulin (cyan) and GABBR1 (cilia base; magenta) and treated or not with 100 nM GLP-1 for 15 min. ( H ) Representative TIRF microscopy recordings of Smo-EpacS H187 FRET ratio changes in the cytosol (black) and cilium (magenta) of MIN6 cells exposed to 100 nM GLP-1, 10 μM forskolin, and 100 μM IBMX. ( I ) Difference in half-maximal cAMP increases in the cytosol and cilium in response to forskolin (1 and 10 µM), IBMX (50 µM), a combination of forskolin and IBMX (F + I) or GLP-1 (100 nM) (means ± SEM; n = 11, 22, 16, 12, and 17 cells from three to six experiments). Statistical significance was assessed with one-sample t test. ( J ) Means of ±SEM for the Smo-EpacS H187 FRET ratio change in the cytosol (black) and cilia (magenta) in response to Forskolin, IBMX, forskolin+IBMX or GLP-1 (two-tailed paired Student´s t test).

Article Snippet: Rabbit anti-GABBR1 (1:200) , Alomone labs , AGB001AN102.

Techniques: Microscopy, Expressing, Fluorescence, Two Tailed Test, Confocal Microscopy

Journal: The EMBO Journal

Article Title: Somatostatin triggers local cAMP and Ca 2+ signaling in primary cilia to modulate pancreatic β-cell function

doi: 10.1038/s44318-025-00383-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-GABBR1 (1:200) , Alomone labs , AGB001AN102.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Virus, Cloning, Modification, Software, Microscopy, Real-time Polymerase Chain Reaction, SYBR Green Assay, Ligation