Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: GPSM3 transcript abundance in whole blood from individuals homozygous for the minor allele of the rs204989/rs204991 haploblock (the “m/m” genotype) is significantly lower than in whole blood from individuals homozygous for the major allele of the haploblock (the “M/M” genotype). ( A ) Schematic representation of identified polymorphisms with respect to the predicted transcription start site of the human GPSM3 gene. ( B ) Scatter plot representation of GPSM3 mRNA abundance, measured in whole blood by qRT-PCR, as normalized to the average level measured in M/M individuals. Individual results for homozygous major allele samples (M/M; n = 53) are depicted by solid circles, and homozygous minor allele samples (m/m; n=11) are depicted by open circles. An average decrease (Δμ) in GPSM3 transcript abundance of 24.1% (t = 3.80, df = 62, p = 0.0003) was observed in homozygous minor allele samples. Error bars represent S.E.M. ( C ) Descriptive statistics of the three cohorts of volunteers genotyped in this study: Minor allele frequencies were 23% for rs204989 and rs204991 in the rheumatoid arthritis samples versus 18% in the disease-free controls (Fisher's exact test; p = 0.4839). Risk/minor allele frequency for rs2812378 was 30.0% in the rheumatoid arthris samples versus 25.0% in the disease-free controls (p = 0.5267). Risk/minor allele frequency for the HLA gene region biallelic SNP rs6457620 was 35.0% in the rheumatoid arthritis samples and 45.0% in the disease-free matched controls (p = 0.1938).

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Quantitative RT-PCR

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Linkage of the GPSM3 SNP haploblock and the known RA risk allele in the HLA gene region, rs6457620 [C>G], is more pronounced in RA samples than controls, but this linkage does not result in a rs6457620 genotype-dependent effect on GPSM3 transcript abundance in whole blood. ( A ) Descriptive statistics of all 200 volunteers, including the RA (n = 50), disease-free matched control (n = 50), and young healthy control volunteers recruited for the current study. These data are stratified by GPSM3 SNP haplotype, showing that the rs6457620 [G] risk allele frequency amongst homozygous ancestral GPSM3 haploblock individuals (M/M) is 41.3%, heterozygous individuals (M/m) is 56.3%, and homozygous minor GPSM3 haploblock (m/m) is 60.7%. These data are significantly different from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0066). ( B ) Analyses of GPSM3 SNP haploblock linkage within RA samples stratified by rs6457620 genotype, again exhibiting statistically significant difference from chance distribution (Fisher-Freeman-Halton exact test; p = 0.0123). ( C ) Within the disease-free matched control samples, GPSM3 SNP minor allele (m) frequency, as stratified by rs6457620 genotype, is not significantly different from chance distribution as determined by Fisher-Freeman-Halton exact test (p = 0.2739). ( D ) Graphical representation of minor allele frequencies (MAFs; of European [‘eu’] population), physical distance (in basepairs), and linkage disequilibrium (LD) value (as quantified by heatmap) between the three linked SNPs of the GPSM3 SNP haploblock (rs204989, rs204990, rs204991) and the HLA gene region SNP rs6547620, as obtained using the NIEHS SNPinfo webserver ( http://snpinfo.niehs.nih.gov/ ) from NCBI dbSNP data (ref. ). ( E ) Despite modest linkage between GPSM3 SNP minor alleles (m) and the rs6457620 risk allele (G), there is no effect of the genotype at HLA gene region SNP rs6457620 on GPSM3 transcript abundance. Significance determined by one-way ANOVA with Bonferroni post-hoc .

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Control

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Functional consequences of SNPs rs204989, rs204990, rs204991, and rs3096688 on GPSM3 promoter activity. ( A ) Functional results upon subcloning a 3.5 kb-region immediately 5′ to the GPSM3 transcription start site from M/M and m/m genotype volunteers, leading to wild type (pGL3-M) and “minor”/polymorphic (pGL3-m) promoter-driven expression of firefly luciferase in HEK293T cells. Luciferase activity is reported as a ratio of GPSM3 promoter-dependent firefly luciferase activity (FLuc) to control Renilla luciferase activity (pRL-TK control vector) and normalized to the average level measured from the wild type pGL3-M vector (set to 1.00; dotted line). Introduction of independent minor alleles (denoted “+###”) into the wild type pGL3-M vector is seen to differentially affect resultant firefly luciferase activity. ( B ) Restoration of the major allele at rs204989 in the pGL3-m vector ( i.e. , “pGL-m-989”) restores wild type GPSM3 promoter activity. All data are compiled from three independent experiments. Error measure is S.E.M. Significance was determined using one-way ANOVA after controlling for multiple comparisons with Dunnett's test using pGL3-M as the defined control.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Functional Assay, Activity Assay, Subcloning, Expressing, Luciferase, Control, Plasmid Preparation

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Putative transcription factor binding sites within the human GPSM3 promoter potentially disrupted by the minor allele of SNP rs204989 (arrows), as predicted by the JASPAR database: ( A ) Fos/Jun (AP1) heterodimer; (B) C/EBP-β.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: Binding Assay

Journal: Genes and immunity

Article Title: Genetic variations in GPSM3 associated with protection from rheumatoid arthritis affect its transcript abundance

doi: 10.1038/gene.2016.3

Figure Lengend Snippet: Lentiviral-mediated shRNA knockdown of endogenous GPSM3 expression in the human monocytic THP-1 cell line disrupts migration toward MCP-1 ( A ) Western blot showing whole lysate (40 μg) immunoreactivity with mouse monoclonal anti-GPSM3 antibody 35.5.1 (ref. ) and with anti-β-actin as a loading control. ( B ) Real-time Transwell migration analysis of indicated calcein-AM stained THP-1 cell lines as shown by change in the ratio of MCP-1-induced to vehicle-induced relative fluorescence units (RFU) over a 30 minute timeframe. ( C ) qRT-PCR data from indicated stable knockdown or scrambled control THP-1 cell line total RNA preparations. There was no significant difference in CCR2 mRNA abundance in the THP-1 cell lines as determined by one-way ANOVA (F = 2.40; p = 0.119). ( D ) Regression analysis-derived rate of THP-1 cell line transmigration over initial migratory period; differences between each cell line was calculated by one-way ANOVA. ( E ) Regression analysis-derived maximum THP-1 cell transmigration over 30 minute timecourese; differences between each cell line were calculated by one-way ANOVA. All data are reported as means with error bars representing S.E.M.

Article Snippet: SNP genotypes were determined using the Type-it Fast Probe PCR Kit (Qiagen), TaqMan SNP genotyping probes, and primers for rs204989 ( GPSM3 ), rs204991 ( GPSM3 ), rs2812378 ( CCL21 ), and rs6457620 ( HLA gene region) (Life Technologies; cat. #C_3293828_20, C_2412452_20, C_16113556_10, and C_29315329_10, respectively) using the manufacturer's suggested protocols.

Techniques: shRNA, Knockdown, Expressing, Migration, Western Blot, Control, Staining, Fluorescence, Quantitative RT-PCR, Derivative Assay, Transmigration Assay

Journal: The European journal of neuroscience

Article Title: Transient viral-mediated overexpression of α-calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell leads to long-lasting functional upregulation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors: dopamine type-1 receptor and protein kinase A dependence

doi: 10.1111/j.1460-9568.2010.07155.x

Figure Lengend Snippet: Location of microinjection cannula tips in the NAcc shell for all rats included in the data analyses. Line drawings of coronal sections (adapted from Paxinos and Watson, 1997) show the location of the microinjection cannula tips in the NAcc shell for rats tested with no inhibitor (A), with SCH23390 (B) or with Rp-cAMPS (C). Numbers to the right indicate mm from bregma. Symbols denote group affiliation: filled circles, αCaMKII-AMPA; open circles, control-AMPA; filled squares, αCaMKII-saline; open ssquares, control-saline.

Article Snippet: Rats in separate groups were administered saline (0.5 µL/side) or the AMPA receptor agonist (±) AMPA hydrobromide (0.8 nmol/0.5 µL/side; Sigma-Aldrich Inc.) infused either alone or with the D1 receptor antagonist SCH23390 HCl (0.8 nmol/0.5 µL/side; Tocris Bioscience, Ellisville, MO, USA) or the PKA inhibitor Rp-cAMPS (80 nmol/1 µL/side; Axxora LLC, San Diego, CA, USA).

Techniques:

Journal: The European journal of neuroscience

Article Title: Transient viral-mediated overexpression of α-calcium/calmodulin-dependent protein kinase II in the nucleus accumbens shell leads to long-lasting functional upregulation of α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors: dopamine type-1 receptor and protein kinase A dependence

doi: 10.1111/j.1460-9568.2010.07155.x

Figure Lengend Snippet: Enhanced locomotor responding to NAcc shell AMPA at 20 days post-infection requires D1 receptor and PKA activation. Data in (A–F) are shown as group mean (±SEM) locomotor counts obtained before and after the challenge injection (arrows). (A and B) The locomotor response to NAcc shell AMPA was significantly enhanced in αCaMKII compared with control group rats. Little locomotor activity was observed in either group following NAcc shell saline. Coadministration of either SCH23390 (C and D) or Rp-cAMPS (E and F) with AMPA into the NAcc shell blocked the enhanced locomotor response in αCaMKII compared with control group rats but spared the locomotor effects of AMPA in both groups compared with NAcc shell saline. Neither SCH23390 nor Rp-cAMPS produced effects that differed significantly from saline when administered alone. (G) Summary of post-challenge injection results illustrated as group mean (+SEM) 2 h total locomotor counts. *P < 0.001, AMPA vs. saline; †P < 0.01, αCaMKII vs. control; revealed by post-hoc Scheffé comparisons following anova.

Article Snippet: Rats in separate groups were administered saline (0.5 µL/side) or the AMPA receptor agonist (±) AMPA hydrobromide (0.8 nmol/0.5 µL/side; Sigma-Aldrich Inc.) infused either alone or with the D1 receptor antagonist SCH23390 HCl (0.8 nmol/0.5 µL/side; Tocris Bioscience, Ellisville, MO, USA) or the PKA inhibitor Rp-cAMPS (80 nmol/1 µL/side; Axxora LLC, San Diego, CA, USA).

Techniques: Infection, Activation Assay, Injection, Activity Assay, Produced

Journal: Journal of molecular histology

Article Title: Localization and expression profile of Group I and II Activators of G-protein Signaling in the kidney

doi: 10.1007/s10735-014-9605-0

Figure Lengend Snippet: Localization of AGS proteins in normal mouse and rat kidneys

Article Snippet: Gpsm3 , AGS4, G18, NG1 , {"type":"entrez-nucleotide","attrs":{"text":"NM_001003974","term_id":"56961638","term_text":"NM_001003974"}} NM_001003974 , Rn01525971_g1.

Techniques:

Journal: Journal of molecular histology

Article Title: Localization and expression profile of Group I and II Activators of G-protein Signaling in the kidney

doi: 10.1007/s10735-014-9605-0

Figure Lengend Snippet: Localization of Group I and II AGS proteins in normal mouse or rat kidneys. Immunohistochemistry was performed using antigen-specific antibodies to Group I (AGS1/RasD1) and Group II (AGS3/GPSM1, AGS4/GPSM3. AGS5/GPSM2, and AGS6/RGS12) in paraffin-embedded mouse or rat kidneys. All AGS proteins were observed by the brown DAB staining in each respective section. AGS1 antibody (A, B) was used to immunostain mouse kidneys, and antibodies targeted to AGS3 (C), AGS5 (E, F), and AGS6 (G, H) were used to immunostain Sprague Dawley rat kidneys. For AGS1, the sections were co-labeled with Dolichos biflorus agglutinin (DBA), which is a selective marker for collecting ducts. Serial sections were performed using AGS3 (Fig. 1C) and PVE-A (Fig. 1D), a marker of proximal tubules, to demonstrate the selectivity of the AGS3-positive tubules were not proximal tubules, but distal tubular in its expression sites. Glm = glomerulus, BV = blood vessel, arrowhead points to collecting duct tubules (in H); and * indicates proximal tubules (in H). Scale bar = 200 um (A–D); 100 um (E–H).

Article Snippet: Gpsm3 , AGS4, G18, NG1 , {"type":"entrez-nucleotide","attrs":{"text":"NM_001003974","term_id":"56961638","term_text":"NM_001003974"}} NM_001003974 , Rn01525971_g1.

Techniques: Immunohistochemistry, Staining, Labeling, Marker, Expressing

Journal: Journal of molecular histology

Article Title: Localization and expression profile of Group I and II Activators of G-protein Signaling in the kidney

doi: 10.1007/s10735-014-9605-0

Figure Lengend Snippet: AGS gene primers used in quantitative real-time PCR

Article Snippet: Gpsm3 , AGS4, G18, NG1 , {"type":"entrez-nucleotide","attrs":{"text":"NM_001003974","term_id":"56961638","term_text":"NM_001003974"}} NM_001003974 , Rn01525971_g1.

Techniques: