Journal: bioRxiv

Article Title: Gz Enhanced Signal Transduction assaY (G Z ESTY) for GPCR deorphanization

doi: 10.1101/2024.07.26.605282

Figure Lengend Snippet: ( A ) Real-time analysis of ligand-mediated activation of a battery of G i/o/z -PCRs (red) and G s -PCR (green) in cells co-transfected with indicated receptors and the 3-in-one G z ESTY plasmid in the presence of 50 µM IBMX. Cells transfected only with the 3-in-one plasmid served as control (white). The following agonists were applied at 4 minutes: 10 µM dopamine (D2R, D1R), 100 µM clonidine (ADRA2A, ADRA2B, ADRA2C), 10 µM GABA (GABABR), 10 µM DAMGO (MOR), 1 µM SNC-80 (DOR), 10 µM salvinorin A (KOR), 10 µM human galanin (1-30) (GAL1R), 100 µM serotonin (5-HT1B), 10 µM human neuropeptide Y (13-36) (NPYR1), 10 µM lysophosphatidic acid (LPAR2), 10 µM 2-arachidonoyl glycerol (CB1R, CB2R), 10 µM N-formyl-met-leu-phe (FPR1), 1 mM isobutyric acid (FFAR3), 10 µM somatostatin 14 (SSTR1), 10 µM neuropeptide FF (NPFFR1), 10 µM SEW2871 (S1PR1), 10 µM histamine (HRH3), 10 µM quinpirole (D3R), 10 µM TFLLR (PAR1), 10 µM SLIGKV (PAR2), 10 µM MK-6892 (HCA2R), 1 µM teriparatide PTH (PTH1R), 10 µM AB-MECA (ADORA2B), 10 µM NDP-α-MSH (MC4R), and 10 µM CGRP (CLR). Data are shown as means ± SEM; N=5. ( B ) GPCRs that couple to G s and/or G i/o/z and can potentially be detected by GZESTY are highlighted and include 213 out of 249 total ligand-activated GPCRs (86%) (adapted from GPRCdb.org ). ( C ) Quantification of agonist-induced activity for 24 G i/o/z -coupled GPCRs. On the right, the response to agonist of the last seven GPCRs is also reported with a different scale. Data are shown as means ± SEM of the fold change obtained; N=5.

Article Snippet: The following chemicals were purchased: clonidine (Tocris), dopamine (Tocris), GABA (Tocris), serotonin (Tocris), 1-oleoyl lysophosphatidic acid (Tocris), DAMGO (MedChemExpress), TFLLR (MedChemExpress), human PAR-2 (1-6, SLIGKV) (MedChemExpress), human galanin (1-30) (MedChemExpress), IBMX (MedChemExpress), SEW2871 (MedChemExpress), somatostatin-14 (Cpc Scientific), human neuropeptide Y (13-36) (Cpc Scientific), neuropeptide FF (Thermo Scientific Chemicals), MK-6892 (MedChemExpress), 2-arachidonoyl glycerol (Cayman Chemicals), N-Formyl-Met-Leu-Phe (R&D systems), SNC80 (Adipogen), isobutyric acid (TCI chemicals), salvinorin A (ChromaDex Inc.), morphine (Mallinckrodt Chemical Company), human β-endorphin (Sigma-Aldrich), teriparatide (MedChemExpress), NDP-α-MSH (Phoenix Pharmaceuticals), AB-MECA (MedChemExpress), quinpirole (Tocris), calcitonin gene-related peptide (CGRP) (AnaSpec).

Techniques: Activation Assay, Battery, Transfection, Plasmid Preparation, Control, Activity Assay

Journal: NPJ biofilms and microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling.

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: Fig. 2 Liver transcriptome analysis of L. reuteri-treated darkness rats. Heatmaps displaying 36 highly expressed genes (a) and 76 lowly expressed genes (b) in the liver of darkness rats compared with control and DL.reuteri rats through DEG analysis using Ballgown software (| fold change | > 0.6 in the log2 ratio value, raw P < 0.05). c Top terms from GO analysis (above) and terms from KEGG analysis (below) of the 112 differential genes in the DEG analysis. The P values of GO and KEGG analyses were determined on the DAVID website. d Visualization of the top GO and KEGG terms related with lipid metabolism and circadian rhythm in the DEG analysis. e Spearman rank correlations between module eigengenes (ME) and clinical biochemical index in the WGCNA analysis (|ρ | > 0.3, *P < 0.05). f Visualization of the top GO and KEGG terms related with lipid metabolism and circadian rhythm of purple module genes (102) in the WGCNA analysis. Circle, genes; Square, KEGG terms; Hexagon, GO terms. The bigger the square or hexagon, the more genes involved. g Crosstalk among different groups of genes identified the possible target genes taking essential roles in the lipid metabolism of L. reuteri-treated darkness rats. h mRNA abundances of Galr1, Galr2, Nr1d1, Nr1d2, Insig2, Srebf1, Lxra, and Rxra in rat liver detected by qPCR. β-Actin was used as a loading control for qPCR analyses. i Serum galanin concentration detected by ELISA. Statistical analysis (h, i) was performed with one-way ANOVA followed by Newman–Keuls multiple comparison test. n = 8 per group. Data present means ± SEM. *P < 0.05, **P < 0.01.

Article Snippet: Right, immunoreactive bands were densitometrically quantified. b, d mRNA abundance detected by qPCR was presented. e mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after galanin treatment (#1179, Tocris Bioscience, Bristol, UK) at the concentration of 0, 50, 150, and 300 pg/mL for 24 h in HepG2 cells.

Techniques: Control, Software, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: NPJ biofilms and microbiomes

Article Title: Alleviation of Limosilactobacillus reuteri in polycystic ovary syndrome protects against circadian dysrhythmia-induced dyslipidemia via capric acid and GALR1 signaling.

doi: 10.1038/s41522-023-00415-2

Figure Lengend Snippet: Fig. 8 Proposed mechanisms for the amelioration of L. reuteri on dyslipidemia in circadian dysrhythmia-induced PCOS-like rats. Left, circadian dysrhythmia due to constant darkness resulted in dyslipidemia and reproductive hallmarks of PCOS in rats. Elevated galanin-GALR1 induced by darkness exposure functioned as an upstream factor of PI3K/AKT pathway and further suppressed NR1D1-induced SREBF1 transcription and translation, thus inducing hepatic lipid accumulation in PCOS-like rats. Right, L. reuteri supplementation ameliorated dyslipidemia and reproductive hallmarks in circadian dysrhythmia-induced PCOS-like rats. L. reuteri restructured microbiome-metabolome network in darkness rats ameliorating the abundance of Lactobacillus, Clostridium sensu stricto 1, Ruminococcaceae UCG-010, and Family XIII AD3011 group, followed by varied serum levels of cortisol, cis-9-palmitoleic acid, 13-methylmyristic acid, capric acid, and dUMP. Notably, capric acid mediated the inhibition of L. reuteri on hepatic GALR1-PI3K/AKT-NR1D1-SREBP1 pathway, which eventually alleviated dyslipidemia.

Article Snippet: Right, immunoreactive bands were densitometrically quantified. b, d mRNA abundance detected by qPCR was presented. e mRNA and protein abundances of NR1D1, NR1D2, INSIG2, SREBP1, LXR, RXR, P-ERK, T-ERK, P-AKT, and T-AKT after galanin treatment (#1179, Tocris Bioscience, Bristol, UK) at the concentration of 0, 50, 150, and 300 pg/mL for 24 h in HepG2 cells.

Techniques: Inhibition

Journal: The FASEB Journal

Article Title: Long‐term enhancements in antidepressant efficacy and neurogenesis: Effects of intranasal co‐administration of neuropeptide Y 1 receptor (NPY1R) and galanin receptor 2 (GALR2) agonists in the ventral hippocampus

doi: 10.1096/fj.202400087r

Figure Lengend Snippet: FIGURE 1 Behavioral responses to intranasal administration of GALR2 and NPY Y1 receptor agonists in the forced swimming test (FST). Figure 1 depicts the impact of intranasal administration of the galanin receptor 2 (GALR2) agonist (M1145) and the neuropeptide Y1 (NPY Y1) receptor agonist, both separately and in combination, on rodent behavior during the forced swimming test (FST). The graph illustrates the significant antidepressant-like effects observed 3 weeks after simultaneous administration of M1145 and the Y1R agonist, which were substantially mitigated by the concurrent administration of the GALR2 antagonist, M871. Behavioral metrics, including total time spent immobile (A) and engaged in swimming (B), are quantitatively represented. Data points are expressed as the mean ± SEM for groups comprising six animals each. Statistical significance is denoted as follows: For panel A, *p < .05 indicates significance versus Control and M1145 groups; ★p < .05 versus Y1R agonist and M1145 + Y1R + M871 groups; ***p < .001 versus Control and M1145 groups. For panel B, *p < .05 is significant versus Control and M1145 groups; ★p < .05 versus Y1R agonist and M1145 + Y1R + M871 groups; ***p < .001 versus Control and M1145 groups. These significances are based on one-way ANOVA followed by Newman–Keuls post hoc test. Horizontal and vertical lines above the bars indicate inter-group comparisons. Abbreviations used are as follows: Control (Distilled water); M1145 (Galanin 2 receptor agonist, 132 μg); Y1R (Y1R receptor agonist [Leu31-Pro34]NPY, 132 μg); M1145 + Y1R (Co-administration of M1145 and Y1R); M1145 + Y1R + M871 (Co-administration of M1145, Y1R, and GALR2 antagonist M871, 132 μg).

Article Snippet: Brain slices (30 μm- thick) through the ventral hippocampus were prepared using a Cryostat (HM550, Microm International, Walldorf, Germany) and analyzed for GALR2- Y1R heteroreceptor complexes using in situ PLA.40,61 Specificity tests for anti- GALR2 (1:100, Alomone Lab) and anti- NPYY1R (1:200, sc- 21992, Santa Cruz Biotechnology INC, CA) were previously performed.37,38 The detailed procedure, including antibody incubation and confocal microscopy analysis, is outlined in the supplementary material.

Techniques: Control

Journal: The FASEB Journal

Article Title: Long‐term enhancements in antidepressant efficacy and neurogenesis: Effects of intranasal co‐administration of neuropeptide Y 1 receptor (NPY1R) and galanin receptor 2 (GALR2) agonists in the ventral hippocampus

doi: 10.1096/fj.202400087r

Figure Lengend Snippet: FIGURE 2 In Situ Detection of GALR2/NPYY1R Heteroreceptor Complexes within the Ventral Hippocampal Dentate Gyrus (DG) via Proximity Ligation Assay (PLA). Identification of GALR2/NPYY1R heteroreceptor complexes in the ventral hippocampal dentate gyrus (DG) employing the in situ proximity ligation assay (PLA), a sophisticated method enabling the direct visualization of close-proximity protein–protein interactions. Utilizing highly specific primary antibodies targeting each protein, the assay reveals: (A) Positive PLA signals (illustrated as red dots) predominantly localized within the subgranular zone (SGZ) of the dentate gyrus, marking the boundary between the granule cell layer (GCL) and the polymorphic layer (PL) in the ventral hippocampus. These signals extend into the PL, whereas the molecular layer (ML) is distinguished by blue dots, signifying the absence of PLA signals. The depicted anatomical references align with the Bregma −5.6 mm coordinate from the Paxinos and Watson (1998) stereotaxic guide. (B) A quantitative assessment of the PLA signals in the SGZ was performed by counting the red PLA-positive spots per nucleus in each observed field, with this process conducted by an analyst blinded to the treatment conditions. (C, D) Representative microphotographs display a significant increase in the density of GALR2/ NPYY1R positive red PLA blobs in the Sgz subregion following GAL and Y1R agonist co-injection, compared to the control group. GALR2/ NPYY1R heteroreceptor complexes appear as red PLA blobs (clusters) found in high densities per cell in numerous nerve cells, visualized using confocal laser microscopy. Statistical significance is indicated as follows: *p < .05 versus Control and M1145; ★p < .05 versus Y1R agonist and M1145 + Y1R + M871; ***p < .001 versus Control and M1145, determined by one-way ANOVA followed by Newman–Keuls post hoc test. Vertical lines from the horizontal line above bars indicate inter-group comparisons. Data are presented as mean ± SEM, with four rats per group, in duplicates. White arrows indicate PLA-positive clusters, while DCX+ cells are marked by white arrowheads. Dashed lines delineate the Gcl of the dentate gyrus. Nuclei are stained blue with DAPI. Abbreviations used: aCSF (artificial Cerebrospinal fluid, control); GAL 3 nmol (Galanin 3 nmol); Y1 3nmol (NPY Y1 receptor agonist [Leu31-Pro34]NPY 3 nmol); GAL + Y1 3 nmol (Co-administration of GAL and Y1); GAL + Y1 + M871 3 nmol (Co-administration of GAL, Y1, and GALR2 antagonist M871 3 nmol).

Article Snippet: Brain slices (30 μm- thick) through the ventral hippocampus were prepared using a Cryostat (HM550, Microm International, Walldorf, Germany) and analyzed for GALR2- Y1R heteroreceptor complexes using in situ PLA.40,61 Specificity tests for anti- GALR2 (1:100, Alomone Lab) and anti- NPYY1R (1:200, sc- 21992, Santa Cruz Biotechnology INC, CA) were previously performed.37,38 The detailed procedure, including antibody incubation and confocal microscopy analysis, is outlined in the supplementary material.

Techniques: In Situ, Proximity Ligation Assay, Protein-Protein interactions, Injection, Control, Microscopy, Staining

Journal: The FASEB Journal

Article Title: Long‐term enhancements in antidepressant efficacy and neurogenesis: Effects of intranasal co‐administration of neuropeptide Y 1 receptor (NPY1R) and galanin receptor 2 (GALR2) agonists in the ventral hippocampus

doi: 10.1096/fj.202400087r

Figure Lengend Snippet: FIGURE 3 Cell Proliferation Enhancement in the Ventral Dentate Gyrus Following Intranasal Co-administration of Galanin Receptor 2 and Y1R Agonists. Figure 3 illustrates the effects of intranasal co-administration of Galanin receptor 2 (GALR2) and Y1R agonists on cell proliferation within the ventral dentate gyrus of adult rats. It showcases the findings from proliferating cell nuclear antigen (PCNA) immunolabeling in the ventral hippocampal dentate gyrus after administering Galanin 2 receptor agonist (M1145) and Y1R receptor agonist, either singularly or in combination, with or without the GAL 2 receptor antagonist (M871). (A, D) The majority of PCNA-positive cells are found in the subgranular zone (Sgz) of the dentate gyrus, located at the interface between the granular cell layer (Gcl) and the polymorphic layer (P) in the ventral hippocampus. These cells typically form clusters of 3–4 cells. The anatomical reference point is Bregma: −5.6 mm, according to the Paxinos and Watson stereotaxic atlas (2006). (B) This panel quantifies the total PCNA-immunoreactive (IR) cells in the ventral hippocampal dentate gyrus. Data are presented as mean ± SEM, illustrating differences between groups after administration of Control, M1145, Y1R agonist [Leu31-Pro34]NPY, or the co-administration of both agonists with or without M871. (D) Shows enhanced PCNA immunolabeling in the Sgz of the dentate gyrus following co-administration of M1145 and Y1R agonist compared to the control group (C). Arrows highlight clusters of PCNA-positive nerve cells, with dashed lines delineating the Gcl of the dentate gyrus. (E) The quantification of PCNA-IR cells double-labeled with DCX or GFAP in either control or M1145 + Y1R-administered rats indicates that Y1R-GALR2 specifically targets neuroblasts. Data are represented as mean ± SEM, with ***p < .001 versus control according to Student's unpaired t-test. (F) A representative photomicrograph displays DCX+/PCNA+ cells (indicated by white arrows), DCX−/PCNA+ cells (indicated by white arrowheads), and DCX+/PCNA− cells (marked by white asterisks) in the M1145 and Y1R agonist group. Data are expressed as mean ± SEM. Statistical significance is denoted as follows: *p < .05 versus Control and M1145; ★p < .05 versus Y1R agonist and M1145 + Y1R + M871; ***p < .001 versus Control and M1145, based on one-way ANOVA followed by Newman–Keuls post hoc test. Vertical lines from the horizontal line above bars indicate inter-group comparisons. Each group consisted of four animals. Abbreviations used: Control (Distilled water); M1145 (Galanin 2 receptor agonist, 132 μg); Y1R (Y1R receptor agonist [Leu31-Pro34]NPY, 132 μg); M1145 + Y1R (Co-administration of M1145 and Y1R); M1145 + Y1R + M871 (Co-administration of M1145, Y1R, and GALR2 antagonist M871, 132 μg).

Article Snippet: Brain slices (30 μm- thick) through the ventral hippocampus were prepared using a Cryostat (HM550, Microm International, Walldorf, Germany) and analyzed for GALR2- Y1R heteroreceptor complexes using in situ PLA.40,61 Specificity tests for anti- GALR2 (1:100, Alomone Lab) and anti- NPYY1R (1:200, sc- 21992, Santa Cruz Biotechnology INC, CA) were previously performed.37,38 The detailed procedure, including antibody incubation and confocal microscopy analysis, is outlined in the supplementary material.

Techniques: Immunolabeling, Control, Labeling

Journal: The FASEB Journal

Article Title: Long‐term enhancements in antidepressant efficacy and neurogenesis: Effects of intranasal co‐administration of neuropeptide Y 1 receptor (NPY1R) and galanin receptor 2 (GALR2) agonists in the ventral hippocampus

doi: 10.1096/fj.202400087r

Figure Lengend Snippet: FIGURE 4 Doublecortin (DCX) Expression in the Ventral Dentate Gyrus Following Intranasal Co-administration of Galanin Receptor 2 and Y1R Agonists. Expression of doublecortin (DCX), a neurogenesis marker, in the ventral dentate gyrus after intranasal administration of the Galanin 2 receptor agonist (M1145) and Y1R receptor agonist, separately or in combination, and with or without the GAL 2 receptor antagonist (M871). Panels (A) and (D) highlight DCX-positive cells predominantly located in the subgranular zone (Sgz) of the dentate gyrus, marking the boundary between the granular cell layer (Gcl) and the polymorphic layer (P), based on the Bregma coordinate −5.6 mm as detailed in the Paxinos and Watson stereotaxic atlas (2006). Panel (B) provides a quantitative analysis of total DCX-immunoreactive (IR) cells in the ventral hippocampal dentate gyrus following various treatment protocols. (C, D) The micrograph in this panel shows a noticeable increase in DCX immunolabeling in the ventral hippocampus following intranasal co-administration of M1145 and Y1R agonist compared to the control group. Arrows point to DCX-positive neurons, while dashed lines delineate the Gcl of the dentate gyrus. The data are represented as mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Newman–Keuls post hoc test, with *p < .05 indicating significance versus the rest of the groups. Abbreviations used: Control (Distilled water); M1145 (Galanin 2 receptor agonist, 132 μg); Y1R (Y1R receptor agonist [Leu31-Pro34]NPY, 132 μg); M1145 + Y1R (Co-administration of M1145 and Y1R); M1145 + Y1R + M871 (Co-administration of M1145, Y1R, and GALR2 antagonist M871, 132 μg).

Article Snippet: Brain slices (30 μm- thick) through the ventral hippocampus were prepared using a Cryostat (HM550, Microm International, Walldorf, Germany) and analyzed for GALR2- Y1R heteroreceptor complexes using in situ PLA.40,61 Specificity tests for anti- GALR2 (1:100, Alomone Lab) and anti- NPYY1R (1:200, sc- 21992, Santa Cruz Biotechnology INC, CA) were previously performed.37,38 The detailed procedure, including antibody incubation and confocal microscopy analysis, is outlined in the supplementary material.

Techniques: Expressing, Marker, Immunolabeling, Control

Journal: The FASEB Journal

Article Title: Long‐term enhancements in antidepressant efficacy and neurogenesis: Effects of intranasal co‐administration of neuropeptide Y 1 receptor (NPY1R) and galanin receptor 2 (GALR2) agonists in the ventral hippocampus

doi: 10.1096/fj.202400087r

Figure Lengend Snippet: FIGURE 5 Neuronal differentiation of DCX-positive cells in the ventral hippocampus following intranasal infusion of Y1R and GALR2 agonists. Impact of intranasal administration of Y1R and GALR2 agonists on the neuronal differentiation of DCX-positive cells in the ventral hippocampus, emphasizing alterations in dendritic architecture. Panel (A) classifies DCX-labeled neurons according to their dendritic morphology, identifying proliferative, intermediate, and postmitotic differentiation stages. This classification elucidates the neuronal maturation phases affected by the treatments. Panel (B) offers a quantitative evaluation of the distribution of DCX-labeled neurons across these differentiation stages. Data are presented as the percentage of each category, providing a clear depiction of the shifts in neuronal maturation stages. Statistical significance was determined using two-way ANOVA followed by Newman–Keuls post hoc test, with *p < .05 indicating significance versus Control and M1145 for proliferative cells, and ***p < .001 versus the rest of the groups. For postmitotic cells, *p < .05 versus M1145 + Y1R and ***p < .001 versus Control and M1145. Abbreviations used: Control (Distilled water); M1145 (Galanin 2 receptor agonist, 132 μg); Y1R (Y1R receptor agonist [Leu31-Pro34]NPY, 132 μg); M1145 + Y1R (Co-administration of M1145 and Y1R); M1145 + Y1R + M871 (Co-administration of M1145, Y1R, and GALR2 antagonist M871, 132 μg).

Article Snippet: Brain slices (30 μm- thick) through the ventral hippocampus were prepared using a Cryostat (HM550, Microm International, Walldorf, Germany) and analyzed for GALR2- Y1R heteroreceptor complexes using in situ PLA.40,61 Specificity tests for anti- GALR2 (1:100, Alomone Lab) and anti- NPYY1R (1:200, sc- 21992, Santa Cruz Biotechnology INC, CA) were previously performed.37,38 The detailed procedure, including antibody incubation and confocal microscopy analysis, is outlined in the supplementary material.

Techniques: Labeling, Control

Journal: Scientific reports

Article Title: Involvement of galanin and galanin receptor 1 in nociceptive modulation in the central nucleus of amygdala in normal and neuropathic rats.

doi: 10.1038/s41598-017-13944-6

Figure Lengend Snippet: Figure 1. Effects of intra-CeA injection of galanin on the HWLs to thermal and mechanical stimulation in normal rats. Left HWL: (a) and (c); right HWL: (b) and (d). CeA, the central nucleus of amygdala; HWL, hindpaw withdrawal latency. Data are presented as mean ± S.E.M. Two-way ANOVA. ***P < 0.001.

Article Snippet: After transferred to PVDF membranes (Millipore, MA, USA), the membranes were incubated in blocking solution and then incubated with the polyclonal goat anti-galanin antibody (sc-16411, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), polyclonal goat anti-GalR1 antibody (sc-16216, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), beta-actin antibody (Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C overnight.

Techniques: Injection

Journal: Scientific reports

Article Title: Involvement of galanin and galanin receptor 1 in nociceptive modulation in the central nucleus of amygdala in normal and neuropathic rats.

doi: 10.1038/s41598-017-13944-6

Figure Lengend Snippet: Figure 2. Effects of intra-CeA injection of M40 on the galanin-induced increases in HWLs to thermal and mechanical stimulation in normal rats. Left HWL: (a) and (c); right HWL: (b) and (d). Time = 0 min: intra-CeA injection of 1 nmol of galanin; time = 5 min: intra-CeA injection of 0.1 nmol, 0.25 nmol, 0.5 nmol of M40, or 1 μl of 0.9% saline as a control. Two-way ANOVA, **P < 0.01, ***P < 0.001.

Article Snippet: After transferred to PVDF membranes (Millipore, MA, USA), the membranes were incubated in blocking solution and then incubated with the polyclonal goat anti-galanin antibody (sc-16411, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), polyclonal goat anti-GalR1 antibody (sc-16216, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), beta-actin antibody (Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C overnight.

Techniques: Injection, Saline, Control

Journal: Scientific reports

Article Title: Involvement of galanin and galanin receptor 1 in nociceptive modulation in the central nucleus of amygdala in normal and neuropathic rats.

doi: 10.1038/s41598-017-13944-6

Figure Lengend Snippet: Figure 4. Effects of intra-CeA injection CTC on the galanin-induced antinociception in normal rats. (a) Hot plate test; (b) Randall Selitto test. CTC, chelery thrine chloride, a PKC inhibitor. One-way ANOVA followed Bonferroni test. **P < 0.01 and ***P < 0.001.

Article Snippet: After transferred to PVDF membranes (Millipore, MA, USA), the membranes were incubated in blocking solution and then incubated with the polyclonal goat anti-galanin antibody (sc-16411, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), polyclonal goat anti-GalR1 antibody (sc-16216, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), beta-actin antibody (Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C overnight.

Techniques: Injection, Hot Plate Test, Randall–Selitto Test

Journal: Scientific reports

Article Title: Involvement of galanin and galanin receptor 1 in nociceptive modulation in the central nucleus of amygdala in normal and neuropathic rats.

doi: 10.1038/s41598-017-13944-6

Figure Lengend Snippet: Figure 6. Intra-CeA injection of galanin induced antinociception in rats with neuropathic pain. (a) and (b), hot plate test; (c) and (d), Randall Selitto Test. (a) and (c): left HWL; (b) and (d): right HWL. Two-way ANOVA, ***P < 0.001.

Article Snippet: After transferred to PVDF membranes (Millipore, MA, USA), the membranes were incubated in blocking solution and then incubated with the polyclonal goat anti-galanin antibody (sc-16411, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), polyclonal goat anti-GalR1 antibody (sc-16216, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), beta-actin antibody (Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C overnight.

Techniques: Injection, Hot Plate Test, Randall–Selitto Test

Journal: Scientific reports

Article Title: Involvement of galanin and galanin receptor 1 in nociceptive modulation in the central nucleus of amygdala in normal and neuropathic rats.

doi: 10.1038/s41598-017-13944-6

Figure Lengend Snippet: Figure 7. Influences of neuropathic pain on the expression of galanin mRNA levels and galanin content in CeA. (a) Results from RT-PCR test; (b) and (c), results from western blot test. Student’s t-test (two tails), *P < 0.05.

Article Snippet: After transferred to PVDF membranes (Millipore, MA, USA), the membranes were incubated in blocking solution and then incubated with the polyclonal goat anti-galanin antibody (sc-16411, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), polyclonal goat anti-GalR1 antibody (sc-16216, Santa Cruz Biotechnology, Inc, Santa Cruz, California, USA), beta-actin antibody (Beyotime Institute of Biotechnology, Shanghai, China) at 4 °C overnight.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 regulates injury-related expression of neuropeptides and other genes. ( a–i ) Unlesioned and injured FN of Atf3 +/+ and Atf3 −/− mice were subjected to qPCR analysis after 3 days of injury to quantify mRNA abundance of primers indicated. In wt mice (grey bars), facial nerve injury resulted in induction of Sprr2j ( a ), Vip ( b ), Ngf ( c ), Wnt2b ( d ), Galanin ( e ), Grp ( f ), Timp1 ( g ) and the known ATF3 target gene Hsp27 ( h ). In contrast to wt mice, induction of Sprr2j, Vip, Ngf, Wnt2b, Galanin, Grp and Hsp27 mRNA abundance was reduced upon facial nerve lesion in Atf3 mutant mice (white bars). Timp1 mRNA induction was more pronounced upon ATF3 loss-of-function ( g ). The Vip2 receptor ( Vipr2 ) was downregulated by facial nerve injury in wt and ATF3-deficient mice ( i ). Numbers in bars in ( b ) reflect independent biological replicates for experiments in ( a–i ). ( j–l ) Confirmation of reduced galanin expression in ATF3-deficient mice upon facial nerve injury. Deafferented wt ( j ) and ATF3-deficient ( k ) FN were stained with anti-galanin directed antibodies. In wt mice ( j ), galanin localized in secretory vesicle-like structures (see inset) of FMNs (some labelled with an arrow). The number of galanin immunoreactive FMNs was reduced in Atf3 mutant mice ( k ). ( l ) Quantification of galanin positive neurons without and 3 and 12 days after lesion. Data are presented as mean ± s.d. ** p ≤ 0.01. Scale bar ( j,k ) = 50 µm; inset = 5 µm.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Mutagenesis, Staining

Journal: Open Biology

Article Title: Atf3 mutant mice show reduced axon regeneration and impaired regeneration-associated gene induction after peripheral nerve injury

doi: 10.1098/rsob.160091

Figure Lengend Snippet: ATF3 mediates neuropeptide and Wnt2b expression in primary PNS neurons. ( a–d ) Adult wt or ATF3-deficient mouse DRG neurons were infected with adenoviral (AV) particles resulting in GFP (control) or ATF3 expression. mRNA levels of Atf3 ( a ), Wnt2b ( b ), Galanin ( c ) and Grp ( d ) were analysed by qPCR. Viral infection strongly enhanced Atf3 mRNA abundance in wt and Atf3 mutant neurons ( a ). Wnt2b ( b ), Galanin ( c ) and Grp ( d ) mRNA levels were augmented upon viral ATF3 overexpression in wt and Atf3 mutant DRG neurons. ( e,f ) Primary wt neurons overexpressing GFP or ATF3 were subjected to ChIP analysis with anti-ATF3 or IgG (control) antibodies. ATF3 occupancy at potential ATF3 binding sites of the Galanin ( e ) and Grp ( f ) promoter was tested with qPCR. ATF3 promoter occupancy was observed in ATF3-overexpressing samples only in the presence of anti-ATF3 but not IgG antibodies suggesting ATF3 binding at the Galanin ( e ) and Grp ( f ) promoter. Numbers in bars reflect independent numbers of experiments. Data are presented as mean ± s.d. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.

Article Snippet: Camptothecin was added at 2 μM for 24 h. Recombinant peptides for mouse VIP (Tocris; no.1911), human gastrin releasing peptide (GRP) (Tocris; no.1789), mouse galanin (Tocris; no.2696) and mouse Wnt2b (R&D systems; no.3900-WN-025) were added at 1 nM (VIP, GRP, galanin) and 1 μg ml −1 (Wn2tb) for 24 h to the cultures.

Techniques: Expressing, Infection, Control, Mutagenesis, Over Expression, Binding Assay

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 1. Galanin (Gal) and Gal receptor (Galr)1e3 mRNA expression in nasal mucosa. Gal and Galr1e3 expression levels were analyzed using RT-PCR. In both severe allergic rhinitis (AR) and control mice (Groups D and A, respectively), Gal and Galr2 were present in nasal mucosa (n ¼ 2) and brain tissue samples (n ¼ 1). Hypoxanthine-guanine phosphor- ibosyltransferase (Hprt) was used as a control.

Article Snippet: After washing with TBS-T, the sections were incubated for 30 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech), GAL polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology), an anti-tryptase ε goat antibody (S-13; 1:400; sc-107314; Santa Cruz Biotechnology, Dallas, TX, USA), CD27 antibody (1:100; Bio-Techne, Minneapolis, MN), and anti-mouse IgE antibody (Invitrogen Waltham, MA) for 2 h at room temperature (25 ± 1 C).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 2. Galanin (GAL) and GAL receptor (GALR) 2 protein expression in nasal mucosa. GAL and GALR2 levels were analyzed by western blot and similar between Groups AeD (n ¼ 8/ group). NAK, NaK-ATPase membrane protein.

Article Snippet: After washing with TBS-T, the sections were incubated for 30 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech), GAL polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology), an anti-tryptase ε goat antibody (S-13; 1:400; sc-107314; Santa Cruz Biotechnology, Dallas, TX, USA), CD27 antibody (1:100; Bio-Techne, Minneapolis, MN), and anti-mouse IgE antibody (Invitrogen Waltham, MA) for 2 h at room temperature (25 ± 1 C).

Techniques: Expressing, Western Blot, Membrane

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 3. Galanin (GAL) and GAL receptor (GALR)2 localization in nasal mucosa. (a, b) Representative photomicrograph showing immunohistochemical analysis of GAL and GALR2 localization in nasal mucosa sections in Groups AeD. A, B, C, and D indicate Group A, Group B, Group C, and Group D, respectively. Controls were only stained with (a) anti-goat and (b) anti-rabbit secondary antibody without exposure to primary antibody. Scale bars, 25 mm. (c) Representative photomicrograph showing the results of double staining for GAL (green) and GALR2 (red) in nasal mucosa sections in all groups. GAL and GALR2 were simultaneously visualized in ciliated epithelial (arrows) and submucosal gland cells (ar- rowheads). Scale bars, 50 mm. HE, hematoxylin-eosin.

Article Snippet: After washing with TBS-T, the sections were incubated for 30 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech), GAL polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology), an anti-tryptase ε goat antibody (S-13; 1:400; sc-107314; Santa Cruz Biotechnology, Dallas, TX, USA), CD27 antibody (1:100; Bio-Techne, Minneapolis, MN), and anti-mouse IgE antibody (Invitrogen Waltham, MA) for 2 h at room temperature (25 ± 1 C).

Techniques: Immunohistochemical staining, Staining, Double Staining

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 7. Expression of GALR2 and IgE in B cells present in nasal mucosa after administration of M871, a GALR antagonist. Mice were administered intranasal (IN) PBS after IN PBS administration (PBSþPBS), IN M871 followed by PBS after intraperitoneal (IP) PBS administration (PBSþM871), IN OVA after IP OVA administration (severe ARþOVA), or IN M871 followed by OVA after IP OVA administration (severe ARþM871) on days 28e34. B cells were collected from nasal tissue and the levels of GALR2 (a) and IgE (b) were detected by western blotting.

Article Snippet: After washing with TBS-T, the sections were incubated for 30 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech), GAL polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology), an anti-tryptase ε goat antibody (S-13; 1:400; sc-107314; Santa Cruz Biotechnology, Dallas, TX, USA), CD27 antibody (1:100; Bio-Techne, Minneapolis, MN), and anti-mouse IgE antibody (Invitrogen Waltham, MA) for 2 h at room temperature (25 ± 1 C).

Techniques: Expressing, Western Blot

Journal: Allergology international : official journal of the Japanese Society of Allergology

Article Title: Involvement of galanin and galanin receptor 2 in a mouse model of allergic rhinitis.

doi: 10.1016/j.alit.2021.07.012

Figure Lengend Snippet: Fig. 8. Localization of CD27 and GALR2 in nasal mucosa after administration of M871, a GALR antagonist. Representative photomicrograph showing immunohistochemical analysis of B cell markers, CD27 (green) and GALR2 (red) in nasal mucosa sections after administration of a GALR antagonist. Scale bars ¼ 30 mm.

Article Snippet: After washing with TBS-T, the sections were incubated for 30 min with 5% goat or donkey serum diluted with TBS-T, followed by treatment with a primary GALR2 polyclonal rabbit antibody (1:500; 26459-1-AP; Proteintech), GAL polyclonal goat antibody (1:50; OAEB01591; Aviva Systems Biology), an anti-tryptase ε goat antibody (S-13; 1:400; sc-107314; Santa Cruz Biotechnology, Dallas, TX, USA), CD27 antibody (1:100; Bio-Techne, Minneapolis, MN), and anti-mouse IgE antibody (Invitrogen Waltham, MA) for 2 h at room temperature (25 ± 1 C).

Techniques: Immunohistochemical staining