rabbit anti negr1 antibodies  (Cell Signaling Technology Inc)

Journal: Molecular Psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: A The depression genetic risk SNP rs3101339 is located in the crucial active promoter region of NEGR1 gene across multiple brain tissues. The genetic association between rs3101339 and depression was shown in LocusZoom using our recent results of cross-ancestry GWAS meta-analysis on depression (416,437 cases and 1,308,758 controls). The active promoter is marked with DNase, H3K4me3, H3K27ac, and transcription factors (REST, POLR2A and RAD21). B , C Brain eQTL data from GTEx and ROSMAP showed that rs3101339 is significantly associated with NEGR1 gene expression in caudate, nucleus accumbens, putamen tissues, and aged DLPFC. The risk allele C of rs3101339 is correlated with higher NEGR1 expression. D EMSA showed that the binding capacity of rs3101339-C allele with nuclear proteins extracted from SH-SY5Y cells was stronger than that of rs3101339-A allele. E , F Reporter gene assay showed that the 501 bp DNA sequences containing rs3101339 exhibited stronger promoter activity compared with control vectors, and rs3101339-C allele significantly increased promoter activity compared with rs3101339-A allele in SH-SY5Y and HEK293T cells. G , H Single-base editing assays showed that NEGR1 mRNA expression in HEK293T cells with the rs3101339-AC genotype was significantly higher than that of rs3101339-AA genotype. G Representative Sanger sequencing results of single-cell clones at rs3101339. H The internal reference gene is GAPDH . I The internal reference gene is ACTB . Data represent mean ± SD, n = 5 for E and F , n = 3 for H and I . Statistical analysis was performed using one-way ANOVA test E and F and two-tailed student’s t-test E and F . A P value less than 0.05 was considered as significance. Anterior caudate AC, Dorsolateral prefrontal cortex DLPFC, Hippocampus HIP.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Gene Expression, Expressing, Binding Assay, Reporter Gene Assay, Activity Assay, Control, Sequencing, Clone Assay, Two Tailed Test

Journal: Molecular Psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: A Timeline of test mice model construction and behavioral tests. B Schematic illustration and stereotaxic injection coordinates of control AAVs or NEGR1-OE AAVs in the mPFC of mice (left), representative image showing correct AAV injection site expressing red fluorescent protein (right). C Validation of NEGR1 protein overexpression in the mPFC using western-blot, one representative mouse per group. D Overview of Y maze test. E , F The total number of entering arm and spontaneous alternation (%) in NEGR1-OE (mPFC) mice exhibited no significant difference compared with control mice. G Overview of open field test. H – M The total distance, average speed, times of entering center, average speed in center, distance in corner or center (%) were evaluated during the open field test, showing no significant difference between NEGR1-OE (mPFC) mice and controls. N Overview of elevated plus maze test. O – T The total distance, distance in close arm or open arm (%), average speed in open arm, times of entering open arm, time in open arm were evaluated during the elevated plus maze test, with NEGR1-OE (mPFC) mice exhibiting anxiety-like behaviors indicated by less times of entering open arm compared with controls. U Overview of forced swimming test. V and W Immobility time (%) and struggling time (%) were evaluated during the forced swimming test, showing no difference between NEGR1-OE (mPFC) mice and controls. Data represent mean ± SD, (Control: n = 12 and NEGR1-OE (mPFC): n = 13) for E and F , H – M , O – T , V and W . Two-tailed student’s t-test E , F , H – M , O – T , V , W was used for statistical analysis, with a significance level set at P < 0.05. Anterior cingulate cortex ACC, Prelimbic cortex PL, Infralimbic cortex IL, Caudoputamen CP, Nucleus accumbens ACB.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Injection, Control, Expressing, Biomarker Discovery, Over Expression, Western Blot, Two Tailed Test

Journal: Molecular Psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: A Timeline of the test mice models construction and behavioral tests. B Schematic illustration and stereotaxic injection coordinates of control AAVs or NEGR1-OE AAVs in the vHIP of mice (left), representative image for showing the correct AAV injection site which is expressed red fluorescent protein (right). C The validation of NEGR1 protein overexpression in the vHIP, one representative mouse per group. D Overview of Y maze test. E , F The spontaneous alternation (%) in NEGR1-OE (vHIP) mice was significantly decreased compared with control mice. G Overview of open field test. H – M The total distance, average speed, times of entering center, average speed in center, distance in corner or center (%) were evaluated during the open field test, and NEGR1-OE (vHIP) mice exhibited anxiety-like behaviors with a higher average speed in center and more distance in corner (%) and less distance in center (%) compared with controls. N Overview of elevated plus maze test. O – T The total distance, distance in close arm or open arm (%), average speed in open arm, times of entering open arm, time in open arm were evaluated during the elevated plus maze test, and the NEGR1-OE (vHIP) mice exhibited anxiety-like behaviors at the index of less time of entering open arm (s) compared with controls. U Overview of forced swimming test. V and W The immobility time (%) and struggling time (%) were evaluated during the forced swimming test, and found that the immobility time (%) in NEGR1-OE (vHIP) mice was significantly increased compared with controls, while the struggling time (%) was significantly decreased. Data represent mean ± SD, (Control: n = 12 and NEGR1-OE (vHIP): n = 12) for E and F , H – M , (Control: n = 11 and NEGR1-OE (vHIP): n = 11) for O – T , V and W . Two-tailed student’s t-test E and F , H – M , O – T , V and W was used to test for statistical analysis, P < 0.05 was set as significant level. Hippocampus HIP; Dentate gyrus DG, Cornu Ammonis 1 CA1, Cornu Ammonis 1 CA3, Thalamus TH, Cerebral cortex CTX.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Injection, Control, Biomarker Discovery, Over Expression, Two Tailed Test

Journal: Molecular Psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: A Timeline of the neuronal sparse labeling assay and transmission electron microscopy assay. B Representative images of pyramidal neurons dendritic spines in ventral hippocampus CA1 region from controls and NEGR1-OE (vHIP) mice. C , D Total 40 images (100×) ( n (Control) = 20, n (NEGR1-OE (vHIP)) = 20) from 4 mice (Control: n = 2, NEGR1-OE (vHIP): n = 2) were selected in random for morphological analysis of dendritic spines, total spines, and subtype (stubby, thin and mushroom) spines density were evaluated during neuronal sparse labeling assay, and the total spines density was significantly decreased in NEGR1-OE (vHIP) mice compared to controls in C , and the thin spines and mushroom spines density were also significantly decreased in NEGR1-OE (vHIP) mice compared to controls in D . E Representative images of synaptic ultrastructure in ventral hippocampus CA1 region from controls and NEGR1-OE (vHIP) mice. F Total 68 images (20,000×) ( n (Control) = 33, n (NEGR1-OE (vHIP)) = 35) from 4 mice (Control: n = 2, NEGR1-OE (vHIP): n = 2) were selected in random for analysis typical synapse number per 19 μm 2 . G – K Total 52 images (50,000×) ( n (Control) = 26, n (NEGR1-OE (vHIP)) = 26) from 4 mice (Control: n = 2, NEGR1-OE (vHIP): n = 2) were selected in random for analysis synaptic vesicle density in boutons, synaptic cleft length, PSD thickness, PSD length and PSD area during transmission electron microscopy assay. These results showed that NEGR1-OE (vHIP) mice were significantly decreased in typical synapse number per 19 μm 2 and synaptic vesicle density in boutons compared with controls. Data represent mean ± SD. Two-tailed student’s t-test for C and F – K and two-way ANOVA test followed by FSD for D were used to test for statistical analysis, P < 0.05 was set as significant level.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Labeling, Transmission Assay, Electron Microscopy, Control, Two Tailed Test

Journal: Molecular Psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: A Flow chart of IP-MS for NEGR1 overexpression in the vHIP region of mice, and we identified 64 high confident NEGR1 interacting proteins which the iBAQ ratio of NEGR1_IP to IGG_IP is greater than 5 and unique peptides in NEGR1-IP is greater than 2. B , C Functional analysis was conducted using Cytoscape (3.10.2) with the ClueGo (2.5.10) plug-in, and the 64 high confident NEGR1 interacting proteins were significant enrichment in 14 GO_Biological Processes presented in B , including calcium-ion-regulated exocytosis, synaptic vesicle endocytosis, and calcium ion-regulated exocytosis of neurotransmitters, etc. D Visualization of the protein-protein interaction network for Clathrin-dependent synaptic vesicle endocytosis cluster, and Snap25 seemly play as a central hub in this network.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Protein-Protein interactions, Over Expression, Functional Assay

Journal: Molecular Psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: A The timeline of transcriptomic analysis for NEGR1 overexpression in the vHIP region of mice, and vHIP tissues from test mice (NEGR1-OE (vHIP): n = 3) and control mice ( n = 3) were dissected for RNA sequencing. B A heatmap illustrating the expression levels of ninety-four differentially expressed genes, comprising forty-five upregulated and forty-nine downregulated between NEGR1-OE (vHIP) mice and controls ( P adj < 0.05). C The top 10 terms for each biological process, cellular component, and molecular function enriched by the differentially expressed genes. D Visualization of genes involved in five myelination-related biological processes.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Over Expression, Control, RNA Sequencing, Expressing

Journal: Molecular psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction.

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: Fig. 1 Validation of functional variant rs3101339 in 1p31.1 with its risk allele C upregulating NEGR1 expression. A The depression genetic risk SNP rs3101339 is located in the crucial active promoter region of NEGR1 gene across multiple brain tissues. The genetic association between rs3101339 and depression was shown in LocusZoom using our recent results of cross-ancestry GWAS meta-analysis on depression (416,437 cases and 1,308,758 controls). The active promoter is marked with DNase, H3K4me3, H3K27ac, and transcription factors (REST, POLR2A and RAD21). B, C Brain eQTL data from GTEx and ROSMAP showed that rs3101339 is significantly associated with NEGR1 gene expression in caudate, nucleus accumbens, putamen tissues, and aged DLPFC. The risk allele C of rs3101339 is correlated with higher NEGR1 expression. D EMSA showed that the binding capacity of rs3101339-C allele with nuclear proteins extracted from SH-SY5Y cells was stronger than that of rs3101339-A allele. E, F Reporter gene assay showed that the 501 bp DNA sequences containing rs3101339 exhibited stronger promoter activity compared with control vectors, and rs3101339-C allele significantly increased promoter activity compared with rs3101339-A allele in SH-SY5Y and HEK293T cells. G, H Single-base editing assays showed that NEGR1 mRNA expression in HEK293T cells with the rs3101339-AC genotype was significantly higher than that of rs3101339-AA genotype. G Representative Sanger sequencing results of single- cell clones at rs3101339. H The internal reference gene is GAPDH. I The internal reference gene is ACTB. Data represent mean ± SD, n = 5 for E and F, n = 3 for H and I. Statistical analysis was performed using one-way ANOVA test E and F and two-tailed student’s t-test E and F. A P value less than 0.05 was considered as significance. Anterior caudate AC, Dorsolateral prefrontal cortex DLPFC, Hippocampus HIP.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Biomarker Discovery, Functional Assay, Variant Assay, Expressing, Gene Expression, Binding Assay, Reporter Gene Assay, Activity Assay, Control, Sequencing, Clone Assay, Two Tailed Test

Journal: Molecular psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction.

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: Fig. 2 Overexpressing NEGR1 in the mPFC of mice leads to anxiety-like behaviors. A Timeline of test mice model construction and behavioral tests. B Schematic illustration and stereotaxic injection coordinates of control AAVs or NEGR1-OE AAVs in the mPFC of mice (left), representative image showing correct AAV injection site expressing red fluorescent protein (right). C Validation of NEGR1 protein overexpression in the mPFC using western-blot, one representative mouse per group. D Overview of Y maze test. E, F The total number of entering arm and spontaneous alternation (%) in NEGR1-OE (mPFC) mice exhibited no significant difference compared with control mice. G Overview of open field test. H–M The total distance, average speed, times of entering center, average speed in center, distance in corner or center (%) were evaluated during the open field test, showing no significant difference between NEGR1-OE (mPFC) mice and controls. N Overview of elevated plus maze test. O–T The total distance, distance in close arm or open arm (%), average speed in open arm, times of entering open arm, time in open arm were evaluated during the elevated plus maze test, with NEGR1-OE (mPFC) mice exhibiting anxiety-like behaviors indicated by less times of entering open arm compared with controls. U Overview of forced swimming test. V and W Immobility time (%) and struggling time (%) were evaluated during the forced swimming test, showing no difference between NEGR1-OE (mPFC) mice and controls. Data represent mean ± SD, (Control: n = 12 and NEGR1-OE (mPFC): n = 13) for E and F, H–M, O–T, V and W. Two-tailed student’s t-test E, F, H–M, O–T, V, W was used for statistical analysis, with a significance level set at P < 0.05. Anterior cingulate cortex ACC, Prelimbic cortex PL, Infralimbic cortex IL, Caudoputamen CP, Nucleus accumbens ACB.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Injection, Control, Expressing, Biomarker Discovery, Over Expression, Western Blot, Two Tailed Test

Journal: Molecular psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction.

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: Fig. 3 Overexpressing NEGR1 in the vHIP of mice leads to working memory impairment, anxiety- and depression-like behaviors. A Timeline of the test mice models construction and behavioral tests. B Schematic illustration and stereotaxic injection coordinates of control AAVs or NEGR1-OE AAVs in the vHIP of mice (left), representative image for showing the correct AAV injection site which is expressed red fluorescent protein (right). C The validation of NEGR1 protein overexpression in the vHIP, one representative mouse per group. D Overview of Y maze test. E, F The spontaneous alternation (%) in NEGR1-OE (vHIP) mice was significantly decreased compared with control mice. G Overview of open field test. H–M The total distance, average speed, times of entering center, average speed in center, distance in corner or center (%) were evaluated during the open field test, and NEGR1-OE (vHIP) mice exhibited anxiety-like behaviors with a higher average speed in center and more distance in corner (%) and less distance in center (%) compared with controls. N Overview of elevated plus maze test. O–T The total distance, distance in close arm or open arm (%), average speed in open arm, times of entering open arm, time in open arm were evaluated during the elevated plus maze test, and the NEGR1-OE (vHIP) mice exhibited anxiety-like behaviors at the index of less time of entering open arm (s) compared with controls. U Overview of forced swimming test. V and W The immobility time (%) and struggling time (%) were evaluated during the forced swimming test, and found that the immobility time (%) in NEGR1-OE (vHIP) mice was significantly increased compared with controls, while the struggling time (%) was significantly decreased. Data represent mean ± SD, (Control: n = 12 and NEGR1-OE (vHIP): n = 12) for E and F, H–M, (Control: n = 11 and NEGR1-OE (vHIP): n = 11) for O–T, V and W. Two-tailed student’s t-test E and F, H–M, O–T, V and W was used to test for statistical analysis, P < 0.05 was set as significant level. Hippocampus HIP; Dentate gyrus DG, Cornu Ammonis 1 CA1, Cornu Ammonis 1 CA3, Thalamus TH, Cerebral cortex CTX.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Injection, Control, Biomarker Discovery, Over Expression, Two Tailed Test

Journal: Molecular psychiatry

Article Title: Elevated NEGR1 in brain induces anxiety or depression-like phenotypes and synaptic dysfunction.

doi: 10.1038/s41380-025-03052-7

Figure Lengend Snippet: Fig. 4 Overexpressing NEGR1 in the vHIP of mice induces to the density of spines decrease and synaptic ultrastructure abnormality. A Timeline of the neuronal sparse labeling assay and transmission electron microscopy assay. B Representative images of pyramidal neurons dendritic spines in ventral hippocampus CA1 region from controls and NEGR1-OE (vHIP) mice. C, D Total 40 images (100×) (n (Control) = 20, n (NEGR1-OE (vHIP)) = 20) from 4 mice (Control: n = 2, NEGR1-OE (vHIP): n = 2) were selected in random for morphological analysis of dendritic spines, total spines, and subtype (stubby, thin and mushroom) spines density were evaluated during neuronal sparse labeling assay, and the total spines density was significantly decreased in NEGR1-OE (vHIP) mice compared to controls in C, and the thin spines and mushroom spines density were also significantly decreased in NEGR1-OE (vHIP) mice compared to controls in D. E Representative images of synaptic ultrastructure in ventral hippocampus CA1 region from controls and NEGR1-OE (vHIP) mice. F Total 68 images (20,000×) (n (Control) = 33, n (NEGR1-OE (vHIP)) = 35) from 4 mice (Control: n = 2, NEGR1-OE (vHIP): n = 2) were selected in random for analysis typical synapse number per 19 μm2. G–K Total 52 images (50,000×) (n (Control) = 26, n (NEGR1-OE (vHIP)) = 26) from 4 mice (Control: n = 2, NEGR1-OE (vHIP): n = 2) were selected in random for analysis synaptic vesicle density in boutons, synaptic cleft length, PSD thickness, PSD length and PSD area during transmission electron microscopy assay. These results showed that NEGR1-OE (vHIP) mice were significantly decreased in typical synapse number per 19 μm2 and synaptic vesicle density in boutons compared with controls. Data represent mean ± SD. Two-tailed student’s t-test for C and F–K and two-way ANOVA test followed by FSD for D were used to test for statistical analysis, P < 0.05 was set as significant level.

Article Snippet: Rabbit IgG (10 μL, #2729S, Cell Signaling Technology) and Rabbit anti-NEGR1 antibodies (10 μL, #13674-1-AP, proteintech) were respectively added into the tubes and incubated overnight at 4 °C.

Techniques: Labeling, Transmission Assay, Electron Microscopy, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: A. Generation of Negr1 knockout mice. Schematic diagram of the targeting vector, the wild-type and mutant alleles. Only relevant restriction sites are shown. The positions of the external southern blot probe, as well as the PCR primers, are indicated by asterisks and arrows, respectively. B. Genomic southern blot using the restriction enzymes EcoRI and SpeI. Bands resulting from introduction of a new EcoRI site and deletion of a SpeI site are indicated by asterisks. C. PCR genotyping of transgenic mice wild-type (+/+), heterozygous (+/−) and homozygous (−/−) mice. D. Western blot probed with antibodies specific to NEGR1 and β-actin showing complete loss of NEGR1 protein in knockout (−/−) mice. E. Sequencing of genomic DNA reveals a T-A mutation that converts an isoleucine residue to asparagine at position 87 (I87N). F. Western blots of brain lysate from Negr1 -I87N mice probed with an anti-NEGR1 antibody. G. NEGR1-immunoblotting of NSC-34 cell lysates overexpressing NEGR1-WT, NEGR1-I87N, and mock-transfected cells. H–I. The Negr1 -I87N mutation causes ER retention of NEGR1. Confocal images showing NSC-34 cells co-expressing NEGR1-WT (H) or NEGR1-I87N (I) together with DsRed-ER. NEGR1-WT is predominantly localized at the plasma membrane whereas distribution of NEGR1-I87N clearly overlaps with the DsRed-fluorophore-labeled ER. Nuclei are visualised by DAPI. Scale: 10 µm.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques: Knock-Out, Plasmid Preparation, Mutagenesis, Southern Blot, Transgenic Assay, Western Blot, Sequencing, Transfection, Expressing, Labeling

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: Growth curve of Negr1 -KO females ( Negr1 +/+ , n ∼13; Negr1 +/− , n ∼7; Negr1 −/− , n ∼10), and males ( Negr1 +/+ , n ∼17; Negr1 +/− , n ∼21; Negr1 −/− , n ∼15) on standard chow diet measured across 10 weeks. Data shown are mean +/− SEM within each sex-genotype group at each time point.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques:

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: Repeated measures ANOVA—results for Negr1 -KO experiment.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques:

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: A–D . Body mass (A), Lean mass (B), fat mass (C) of wild type (female, n = 28; male, n = 22), heterozygous (female, n = 52; male, n = 51) and homozygous (female, n = 28; male, n = 24) Negr1 -I87N mice fed on high-fat diet measured across 18 weeks. Data shown are mean +/− SEM within each sex-genotype group at each time point.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques:

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: Repeated measures ANOVA—results for Negr1 -I87N experiment.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques:

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: A–G . Body length (A), body mass (B) and lean mass (C) normalised to body length, liver mass (D) and percentage of liver mass to total body mass (E) of wild-type (female, n = 23; male, n = 16), heterozygous (female, n = 37; male, n = 36) and homozygous (female, n = 17; male, n = 19) Negr1 -I87N mice measured at 22 weeks in females and males. All data are presented as mean ± SEM. Student's t -test was carried out between groups, *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques:

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: A , B . Number of triglyceride droplets classified into ranges of size in diameter (µm) in hepatocytes of female Negr1 -I87N mice (n = 3 mice per genotype). All data are presented as mean ± SEM. Student's t -test (2-tailed) was carried out between groups, *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques:

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: A,B. Food intake over 24 hrs measured at 16-weeks in Negr1 -I87N female (A) (WT, n = 23; hom, n = 20) and male (B) (WT, n = 21; hom, n = 22). All data are presented as mean ± SEM. Student's t -test (2-tailed) was carried out between groups, *, P <0.05; **, P <0.01; ***, P <0.001.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques:

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: A. NEGR1 protein expression in different CNS regions from adult mice. B. Developmental expression regulation of NEGR1 in the postnatal hypothalamus. Blots were probed with antibodies specific to NEGR1 and ubiquitously expressed calnexin (cnx). C. Semiquantitative assessment of hypothalamic NEGR1 protein expression relative to cnx during postnatal development. D–E. Distribution of Negr1 mRNA in the adult murine hypothalamus in reference to bregma (n = 4 mice/condition). Scale bars: 500 µm (D–E); ad: adult; co: cerebral cortex; hy: hypothalamus; hi: hippocampus; cb: cerebellum; bs: brainstem; sc: spinal cord; ob: olfactory bulb; cnx: calnexin; PVN: paraventricular nucleus; DMN: dorsomedial nucleus; VMN: ventromedial nucleus; ARC: arcuate nucleus.

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Functional Inactivation of the Genome-Wide Association Study Obesity Gene Neuronal Growth Regulator 1 in Mice Causes a Body Mass Phenotype

doi: 10.1371/journal.pone.0041537

Figure Lengend Snippet: A–C. NSC-34 cells expressing EGFP alone (control) or together with Negr1 -WT or Negr1 -I87N mutants at 0 min (t1) and following 60 min (t2) of cell aggregation. D. Histogram showing cell aggregation expressed as the ratio of 0 min (t1) and 60 min (t2) time points. EGFP+empty vector (control): 1.06±0.05; NEGR1-WT+EGFP: 2.11±0.2; EGFP+NEGR1-I87N: 0.93±0.2. Data represent means ± sd calculated from three independent experiments. E–G. Confocal images showing hypothalamic neurons immunostained for the neuronal marker βIII-tubulin (red) cultured together with transfected NSC-34 cells (green). NSC34 cells were transfected with (E) pEGFP together with an empty pcDNA-vector (control), (F) pEGFP+pcDNA3-NEGR1-WT and (G) EGFP+NEGR1-I87N. H. Mean neurite lengths of hypothalamic neurons relative to control (set to 100%). Error bars represent SEM from three independent experiments (∼400 neurites per condition). Two-tailed Student's test; * P <0.05; ** P <0.01; *** P <0.001. Scale: 100 µm (A), 20 µm (E).

Article Snippet: The following antibodies were used for immunoblot (IB), immunohistochemistry (IHC) or immunocytochemistry (ICC): mouse anti-β-actin (Sigma, IB, diluted 1∶10.000), rabbit anti-calnexin (Abcam, IB, diluted 1∶1000), rabbit anti-CRH (UCB Bioproducts, IHC, diluted 1∶200), mouse anti-GFP (Millipore, ICC, diluted 1∶1000), rabbit anti-NEGR1 (Sigma, IB, diluted 1∶2000; ICC, diluted 1∶300), rabbit anti-NPY (Immunostar, diluted 1∶2000); rabbit anti-somatostatin (Chemicon, IHC, diluted 1∶1500), rabbit anti-tyrosine hydroxylase (IHC, diluted 1∶2000), mouse anti-βIII tubulin (Covance, ICC, diluted 1∶1000), Cy3- and Alexa 488 Fluor- conjugated secondary antibodies (Molecular Probes, IHC, ICC 1∶500) and HRP-conjugated secondary antibody (Jackson ImmunoResearch, IB 1∶5000). cDNA constructs used in this study: DsRed-ER, pEGFP-C1 (Clontech).

Techniques: Expressing, Plasmid Preparation, Marker, Cell Culture, Transfection, Two Tailed Test