Structured Review

4Gene ten env1
Characterization of the lesser and greater hedgehog tenrec ( E . telfairi and S. setosus ) orthologous ten - <t>env1</t> gene and of its proviral integration site. ( A ) Structure of the ten-env1 gene and evidence for orthology between the lesser and greater hedgehog
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Images

1) Product Images from "Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs"

Article Title: Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1412268111

Characterization of the lesser and greater hedgehog tenrec ( E . telfairi and S. setosus ) orthologous ten - env1 gene and of its proviral integration site. ( A ) Structure of the ten-env1 gene and evidence for orthology between the lesser and greater hedgehog
Figure Legend Snippet: Characterization of the lesser and greater hedgehog tenrec ( E . telfairi and S. setosus ) orthologous ten - env1 gene and of its proviral integration site. ( A ) Structure of the ten-env1 gene and evidence for orthology between the lesser and greater hedgehog

Techniques Used:

2) Product Images from "Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression"

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression

Journal: BMC Immunology

doi: 10.1186/s12865-017-0198-8

Detection of cell surface molecules and signaling pathway molecules after knockdown and over-expression of Foxo1 in Treg cells. a , Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with Foxo1 siRNA by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 siRNA). b Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with over-expression plasmid of Foxo1 by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 over-expression). c Detected of Median Fluorescence Intensity (MFI) for CD127 in Treg cells 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid by flow cytometry. d Representative western blot of p-Erk1/2, total Erk1/2, p-Akt, total Akt, p-Stat5, total Stat5, p-Foxo1 and total Foxo1 in Treg cells 48 h after transfection with Foxo1 siRNA and over-expression plasmid of Foxo1, GADPH was used as a control. Treg cells stimulated with anti-CD3 (0.01 μg/ml) and anti-CD28 (1.0 μg/ml) in medium during culture. e Expression of mRNA for IL-2, IL-4, IL-7 and IL-15 in Treg cells, 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid. All experiments were repeated at least three times. ** P
Figure Legend Snippet: Detection of cell surface molecules and signaling pathway molecules after knockdown and over-expression of Foxo1 in Treg cells. a , Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with Foxo1 siRNA by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 siRNA). b Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with over-expression plasmid of Foxo1 by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 over-expression). c Detected of Median Fluorescence Intensity (MFI) for CD127 in Treg cells 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid by flow cytometry. d Representative western blot of p-Erk1/2, total Erk1/2, p-Akt, total Akt, p-Stat5, total Stat5, p-Foxo1 and total Foxo1 in Treg cells 48 h after transfection with Foxo1 siRNA and over-expression plasmid of Foxo1, GADPH was used as a control. Treg cells stimulated with anti-CD3 (0.01 μg/ml) and anti-CD28 (1.0 μg/ml) in medium during culture. e Expression of mRNA for IL-2, IL-4, IL-7 and IL-15 in Treg cells, 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid. All experiments were repeated at least three times. ** P

Techniques Used: Over Expression, Expressing, Transfection, Flow Cytometry, Cytometry, Plasmid Preparation, Fluorescence, Western Blot

3) Product Images from "Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes"

Article Title: Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

Journal: Eurosurveillance

doi: 10.2807/1560-7917.ES.2018.23.6.17-00672

Multiplex PCR for detection of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 , European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) in the context of animal health and food safety, 2017
Figure Legend Snippet: Multiplex PCR for detection of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 , European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) in the context of animal health and food safety, 2017

Techniques Used: Multiplex Assay, Polymerase Chain Reaction

4) Product Images from "miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation"

Article Title: miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation

Journal: Circulation

doi: 10.1161/CIRCULATIONAHA.117.027799

EVI1 is the novel target of miR-22 in VSMCs. A , The predicted miR-22 binding site within EVI1 3′-UTR by Targetscan. EVI1, miR-22 sequence (mmu-miR-22), and the miR-22 binding site mutant (EVI1 mutant) are depicted in this illustration. The mutation site in EVI1 mutant and corresponding sequences in wild-type EVI1 and mmu-miR-22 are underlined and bold. B , EVI1 was upregulated in the extended cultured VSMCs. Each dot represents EVI1 mRNA level in each passage (P3, P8, P9, P12) normalized to EVI1 mRNA level of P0. C and D , EVI1 was negatively regulated by miR-22. VSMCs transfected with miR-22 mimics, inhibitor, or respective controls (Ctrl), as indicated, were subjected to serum starvation for 48 hours. Total RNA and protein were harvested and subjected to RT-qPCR ( C ) and Western blot ( D ) analyses, respectively. E , miR-22 repressed EVI1 3′-UTR reporter activity. miR-22 mimics, inhibitor, or respective controls (Ctrl) were cotransfected with EVI1 3′-UTR reporter into VSMCs, as indicated. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. F , miR-22 binding site was required for miR-22–mediated EVI1 gene repression. miR-22 mimics or control miR mimics (miRNA ctrl) were cotransfected into VSMCs with wild-type reporter (pmiR-EVI1-WT) or the reporter containing mutated miR-22 binding site (pmiR-EVI1-mutant). Transfected cells were subjected to serum starvation for 48 hours before luciferase activity assay. Data and error bars in B through F are representative ( D ) or mean±SEM ( B , C , E , and F ) (n=4 in D and F ). * P
Figure Legend Snippet: EVI1 is the novel target of miR-22 in VSMCs. A , The predicted miR-22 binding site within EVI1 3′-UTR by Targetscan. EVI1, miR-22 sequence (mmu-miR-22), and the miR-22 binding site mutant (EVI1 mutant) are depicted in this illustration. The mutation site in EVI1 mutant and corresponding sequences in wild-type EVI1 and mmu-miR-22 are underlined and bold. B , EVI1 was upregulated in the extended cultured VSMCs. Each dot represents EVI1 mRNA level in each passage (P3, P8, P9, P12) normalized to EVI1 mRNA level of P0. C and D , EVI1 was negatively regulated by miR-22. VSMCs transfected with miR-22 mimics, inhibitor, or respective controls (Ctrl), as indicated, were subjected to serum starvation for 48 hours. Total RNA and protein were harvested and subjected to RT-qPCR ( C ) and Western blot ( D ) analyses, respectively. E , miR-22 repressed EVI1 3′-UTR reporter activity. miR-22 mimics, inhibitor, or respective controls (Ctrl) were cotransfected with EVI1 3′-UTR reporter into VSMCs, as indicated. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. F , miR-22 binding site was required for miR-22–mediated EVI1 gene repression. miR-22 mimics or control miR mimics (miRNA ctrl) were cotransfected into VSMCs with wild-type reporter (pmiR-EVI1-WT) or the reporter containing mutated miR-22 binding site (pmiR-EVI1-mutant). Transfected cells were subjected to serum starvation for 48 hours before luciferase activity assay. Data and error bars in B through F are representative ( D ) or mean±SEM ( B , C , E , and F ) (n=4 in D and F ). * P

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Cell Culture, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase

EVI1 inhibition reproduces the effects of miR-22 overexpression on VSMC-specific gene expression, proliferation, and migration. A and B , EVI1 knockdown VSMC was generated and validated. Total RNA and protein of control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs were harvested and subjected to RT-qPCR ( A ) and Western blot ( B ) analyses, respectively. C , EVI1 knockdown significantly increases expression of VSMC markers (SMαA, SM22α, CNN1, SM-myh11, SMTN-B) and transcription factors (SRF and Myocd), although the transcription factor MEF2c exhibited no significant change in expression. Total RNA of control and EVI1 stable knockdown VSMCs were harvested and subjected to RT-qPCR. D , Inhibition of endogenous EVI1 decreases VSMC proliferation. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by BrdU incorporation assays in response to no (Ctrl), serum (20%), and PDGF-BB (10 ng/mL) stimulation. E , Inhibition of endogenous EVI1 decreases VSMC migration. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by transwell migration in response to serum (20%) and PDGF-BB (30 ng/mL) stimulation. Note: No or very few migrated cells were observed without cell chemoattractant in transwell migration assays; therefore, no control treatment is shown. Data and error bars are representative ( B ) or mean±SEM ( A and C through E ) (n=3 in C ; 4 in D ; or 5 in E ). * P
Figure Legend Snippet: EVI1 inhibition reproduces the effects of miR-22 overexpression on VSMC-specific gene expression, proliferation, and migration. A and B , EVI1 knockdown VSMC was generated and validated. Total RNA and protein of control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs were harvested and subjected to RT-qPCR ( A ) and Western blot ( B ) analyses, respectively. C , EVI1 knockdown significantly increases expression of VSMC markers (SMαA, SM22α, CNN1, SM-myh11, SMTN-B) and transcription factors (SRF and Myocd), although the transcription factor MEF2c exhibited no significant change in expression. Total RNA of control and EVI1 stable knockdown VSMCs were harvested and subjected to RT-qPCR. D , Inhibition of endogenous EVI1 decreases VSMC proliferation. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by BrdU incorporation assays in response to no (Ctrl), serum (20%), and PDGF-BB (10 ng/mL) stimulation. E , Inhibition of endogenous EVI1 decreases VSMC migration. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by transwell migration in response to serum (20%) and PDGF-BB (30 ng/mL) stimulation. Note: No or very few migrated cells were observed without cell chemoattractant in transwell migration assays; therefore, no control treatment is shown. Data and error bars are representative ( B ) or mean±SEM ( A and C through E ) (n=3 in C ; 4 in D ; or 5 in E ). * P

Techniques Used: Inhibition, Over Expression, Expressing, Migration, Generated, shRNA, Quantitative RT-PCR, Western Blot, BrdU Incorporation Assay

EVI1 functions as a transcriptional repressor for VSMC gene expression. A and B , SRF binding element(s) are required for EVI1-mediated SMαA and SM22α gene promoter activity. Wild-type (SMαA/SM22α) or SRF binding site(s) mutants (SRF mut ) of SMαA ( A ) and SM22α ( B ) gene promoter reporters were transfected into control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. C and D , EVI1 inhibition significantly increases SRF and Myocd gene promoter activity. SRF ( C ) and Myocd ( D ) gene promoter reporters were transfected into control and EVI1 stable knockdown VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. E , EVI1 was significantly enriched at the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes and was significantly decreased by EVI1 knockdown, indicating direct binding of EVI1 to these promoter regions. ChIP assays were performed to measure EVI1 enrichment in the promoter region of its downstream targets. Control (sh-EVI1–) and EVI1 stable knockdown (sh-EVI1+) VSMCs were lysed and incubated with antibody against EVI1 to immunoprecipitate EVI1-bound promoter DNA, followed by qPCR to quantify DNA enrichment. For SMαA and SM22α, genomic DNA without SRF binding sites were amplified as additional control for specific promoter DNA enrichment and designated as Promoter–. For SRF and Myocd, PCR amplification of DNA region adjacent to their promoter was designated as Promoter–. F , H3K9me3 enrichment within the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes was significantly decreased by EVI1 knockdown. ChIP assays were performed by using an antibody against H3K9me3. Data and error bars are mean±SEM. * P
Figure Legend Snippet: EVI1 functions as a transcriptional repressor for VSMC gene expression. A and B , SRF binding element(s) are required for EVI1-mediated SMαA and SM22α gene promoter activity. Wild-type (SMαA/SM22α) or SRF binding site(s) mutants (SRF mut ) of SMαA ( A ) and SM22α ( B ) gene promoter reporters were transfected into control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. C and D , EVI1 inhibition significantly increases SRF and Myocd gene promoter activity. SRF ( C ) and Myocd ( D ) gene promoter reporters were transfected into control and EVI1 stable knockdown VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. E , EVI1 was significantly enriched at the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes and was significantly decreased by EVI1 knockdown, indicating direct binding of EVI1 to these promoter regions. ChIP assays were performed to measure EVI1 enrichment in the promoter region of its downstream targets. Control (sh-EVI1–) and EVI1 stable knockdown (sh-EVI1+) VSMCs were lysed and incubated with antibody against EVI1 to immunoprecipitate EVI1-bound promoter DNA, followed by qPCR to quantify DNA enrichment. For SMαA and SM22α, genomic DNA without SRF binding sites were amplified as additional control for specific promoter DNA enrichment and designated as Promoter–. For SRF and Myocd, PCR amplification of DNA region adjacent to their promoter was designated as Promoter–. F , H3K9me3 enrichment within the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes was significantly decreased by EVI1 knockdown. ChIP assays were performed by using an antibody against H3K9me3. Data and error bars are mean±SEM. * P

Techniques Used: Expressing, Binding Assay, Activity Assay, Transfection, shRNA, Luciferase, Inhibition, Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

5) Product Images from "CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer's disease-like pathogenesis in APP+PS1 bigenic mice"

Article Title: CNS expression of anti-inflammatory cytokine interleukin-4 attenuates Alzheimer's disease-like pathogenesis in APP+PS1 bigenic mice

Journal: The FASEB Journal

doi: 10.1096/fj.10-155317

AAV injection and strategy. A ) AAV-mediated somatic gene transfer of IL-4. Non-Tg mice at 3 mo of age received bilateral hippocampal injection of AAV-GFP (2×10 9 viral particles (VP)/hippocampus, dotted line) or AAV-IL-4 (2×10 9 VP/hippocampus,
Figure Legend Snippet: AAV injection and strategy. A ) AAV-mediated somatic gene transfer of IL-4. Non-Tg mice at 3 mo of age received bilateral hippocampal injection of AAV-GFP (2×10 9 viral particles (VP)/hippocampus, dotted line) or AAV-IL-4 (2×10 9 VP/hippocampus,

Techniques Used: Injection, Mouse Assay

Gene delivery of IL-4 enhances neurogenesis in SGZ of APP+PS1 mice. A – C ) Representative images of Dcx staining in the dentate gyrus of non-Tg ( A ) or APP+PS1 mice injected with AAV-GFP ( B ) or AAV-IL-4 ( C ) into the hippocampus at
Figure Legend Snippet: Gene delivery of IL-4 enhances neurogenesis in SGZ of APP+PS1 mice. A – C ) Representative images of Dcx staining in the dentate gyrus of non-Tg ( A ) or APP+PS1 mice injected with AAV-GFP ( B ) or AAV-IL-4 ( C ) into the hippocampus at

Techniques Used: Mouse Assay, Staining, Injection

Aβ deposition in the hippocampal region of gene-delivered APP+PS1 mouse brain. A – D ) Frozen brain sections of APP+PS1 mice injected with AAV-GFP ( A , B ) or AAV-IL-4 ( C , D ) were immunostained with anti-Aβ antibody
Figure Legend Snippet: Aβ deposition in the hippocampal region of gene-delivered APP+PS1 mouse brain. A – D ) Frozen brain sections of APP+PS1 mice injected with AAV-GFP ( A , B ) or AAV-IL-4 ( C , D ) were immunostained with anti-Aβ antibody

Techniques Used: Mouse Assay, Injection

Gene delivery of IL-4 suppresses glial accumulation in APP+PS1 mice. A – D ) APP+PS1 mice injected with AAV-GFP ( A , B ) or AAV-IL-4 ( C , D ) at 3 mo of age were sacrificed at 8 mo of age. Hippocampal frozen sections were immunostained
Figure Legend Snippet: Gene delivery of IL-4 suppresses glial accumulation in APP+PS1 mice. A – D ) APP+PS1 mice injected with AAV-GFP ( A , B ) or AAV-IL-4 ( C , D ) at 3 mo of age were sacrificed at 8 mo of age. Hippocampal frozen sections were immunostained

Techniques Used: Mouse Assay, Injection

IL-4 promotes the expression and phosphorylation of NR2B. A ) Immunoblotting (top) and quantification (bottom) of NR2A and NR2B in the membrane-enriched fraction of the mouse hippocampus in non-Tg (NTg), uninjected APP+PS1 (uninj.), APP+PS1
Figure Legend Snippet: IL-4 promotes the expression and phosphorylation of NR2B. A ) Immunoblotting (top) and quantification (bottom) of NR2A and NR2B in the membrane-enriched fraction of the mouse hippocampus in non-Tg (NTg), uninjected APP+PS1 (uninj.), APP+PS1

Techniques Used: Expressing

Gene delivery of IL-4 improves memory function of APP+PS1 mice. APP+PS1 mice received bilateral hippocampal injection of AAV-GFP or AAV-IL-4 at 3 mo of age and were tested by the 2-d RAWM task at 7–8 mo of age. Non-Tg and uninjected
Figure Legend Snippet: Gene delivery of IL-4 improves memory function of APP+PS1 mice. APP+PS1 mice received bilateral hippocampal injection of AAV-GFP or AAV-IL-4 at 3 mo of age and were tested by the 2-d RAWM task at 7–8 mo of age. Non-Tg and uninjected

Techniques Used: Mouse Assay, Injection

6) Product Images from "Asp299Gly and Thr399Ile polymorphism of TLR-4 gene in patients with prostate cancer from North India"

Article Title: Asp299Gly and Thr399Ile polymorphism of TLR-4 gene in patients with prostate cancer from North India

Journal: Indian Journal of Urology : IJU : Journal of the Urological Society of India

doi: 10.4103/0970-1591.109982

Agarose gel (2%) showing PCR amplification of TLR-4 (a, b) and restriction digestion of PCR products (c, d). Lanes M:Φ×BsuR1(HaeIII)[NEB]; 5-11 (c) and 1,2,6-9 (d): wild-type allele; 1,2 (c) and 3,4,10 (d) heterozygous alleles; 3,4 (c) and 11 (d): homozygous variant allele
Figure Legend Snippet: Agarose gel (2%) showing PCR amplification of TLR-4 (a, b) and restriction digestion of PCR products (c, d). Lanes M:Φ×BsuR1(HaeIII)[NEB]; 5-11 (c) and 1,2,6-9 (d): wild-type allele; 1,2 (c) and 3,4,10 (d) heterozygous alleles; 3,4 (c) and 11 (d): homozygous variant allele

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Variant Assay

7) Product Images from "Establishment of Immortalized BMP2/4 Double Knock‐Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis"

Article Title: Establishment of Immortalized BMP2/4 Double Knock‐Out Osteoblastic Cells Is Essential for Study of Osteoblast Growth, Differentiation, and Osteogenesis

Journal: Journal of Cellular Physiology

doi: 10.1002/jcp.25266

Generation of an immortalized mouse deleted BMP2/4 osteoblast cell line. (A) Strategy for generation of immortalized BMP2/4 ko/ko osteoblasts. (B) The iBMP2/4 fx/fx ob cells were infected with adenovirus carrying Cre recombinase and GFP genes for 14 h. The GFP positive cells were observed under a Nikon inverted fluorescent microscope. (C and D) Genotyping and PCR strategy. Genomic DNAs from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were isolated and amplified by the BMP2/4 specific primers shown in Table I . (C) primers, a and b, c and d, amplify fragments of 400, 403 bp from iBMP2/4 fx/fx osteoblasts; primers, a and e, amplify fragment of 564 bp from iBMP2/4 ko/ko osteoblasts. (D) primers, f and g, h and i, amplify fragments of 184, 216 bp from iBMP2/4 fx/fx osteoblasts; primers, f and j, amplify fragment of 418 bp from iBMP2/4 ko/ko osteoblasts. The amplified PCR products were run on 1% agarose gels and stained with ethidium bromide. Lane 1, lower molecular DNA marker; lane 2, negative control. Lanes 3 and 4. Genomic DNAs isolated from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were amplified using the floxed BMP2 and BMP4 primers, respectively. Lanes 5 and 6. Genomic DNAs isolated from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were amplified using the recombinant BMP2 and BMP4 primers, respectively. Lanes 7 and 8. Genomic DNAs isolated from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were amplified using the BMP2 exon 3 and BMP4 exon 4 primers, respectively. (E) The iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were photographed under a Nikon inverted microscope. (F) The iBmp2/4 ko/ko ob cells were treated with or without BMP2 (100 ng/ml) plus BMP4 (20 ng/ml) for 48 h and cell morphology was observed under the microscope. (G) Cell length of iBMP2/4 fx/fx , iBMP2/4 ko/ko ob cells, and iBMP2/4 ko/ko ob cells treated with BMP2/4 was quantitated. (H) Morphology of the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells was observed using scanning electron microscope. Fx. floxed; Con, control; Rec, recombinant; Exon, BMP2 exon 3; BMP4 exon 4; ko, knock‐out; n, number. ** P
Figure Legend Snippet: Generation of an immortalized mouse deleted BMP2/4 osteoblast cell line. (A) Strategy for generation of immortalized BMP2/4 ko/ko osteoblasts. (B) The iBMP2/4 fx/fx ob cells were infected with adenovirus carrying Cre recombinase and GFP genes for 14 h. The GFP positive cells were observed under a Nikon inverted fluorescent microscope. (C and D) Genotyping and PCR strategy. Genomic DNAs from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were isolated and amplified by the BMP2/4 specific primers shown in Table I . (C) primers, a and b, c and d, amplify fragments of 400, 403 bp from iBMP2/4 fx/fx osteoblasts; primers, a and e, amplify fragment of 564 bp from iBMP2/4 ko/ko osteoblasts. (D) primers, f and g, h and i, amplify fragments of 184, 216 bp from iBMP2/4 fx/fx osteoblasts; primers, f and j, amplify fragment of 418 bp from iBMP2/4 ko/ko osteoblasts. The amplified PCR products were run on 1% agarose gels and stained with ethidium bromide. Lane 1, lower molecular DNA marker; lane 2, negative control. Lanes 3 and 4. Genomic DNAs isolated from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were amplified using the floxed BMP2 and BMP4 primers, respectively. Lanes 5 and 6. Genomic DNAs isolated from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were amplified using the recombinant BMP2 and BMP4 primers, respectively. Lanes 7 and 8. Genomic DNAs isolated from the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were amplified using the BMP2 exon 3 and BMP4 exon 4 primers, respectively. (E) The iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells were photographed under a Nikon inverted microscope. (F) The iBmp2/4 ko/ko ob cells were treated with or without BMP2 (100 ng/ml) plus BMP4 (20 ng/ml) for 48 h and cell morphology was observed under the microscope. (G) Cell length of iBMP2/4 fx/fx , iBMP2/4 ko/ko ob cells, and iBMP2/4 ko/ko ob cells treated with BMP2/4 was quantitated. (H) Morphology of the iBMP2/4 fx/fx and iBMP2/4 ko/ko ob cells was observed using scanning electron microscope. Fx. floxed; Con, control; Rec, recombinant; Exon, BMP2 exon 3; BMP4 exon 4; ko, knock‐out; n, number. ** P

Techniques Used: Infection, Microscopy, Polymerase Chain Reaction, Isolation, Amplification, Staining, Marker, Negative Control, Recombinant, Inverted Microscopy, Knock-Out

8) Product Images from "Human Natural Killer T Cells Infiltrate into the Skin at Elicitation Sites of Allergic Contact Dermatitis"

Article Title: Human Natural Killer T Cells Infiltrate into the Skin at Elicitation Sites of Allergic Contact Dermatitis

Journal: The Journal of investigative dermatology

doi: 10.1038/sj.jid.5701199

Unique Vα24JαQ NKT-associated TCR gene expression occurs in ACD, but not in normal skin Nested PCR to detect unique Vα24-JαQ TCR sequences in skin biopsy specimens. RNA was extracted from an NKT cell line, Jurkat cells, and foreskin KC (FSKC), cDNA was synthesized, subjected to the nested PCR, and run on an agarose gel as described in Methods. ( a ) Control lane (Ctrl) represents no cDNA added to nested PCR. ( b ) This same nested PCR was applied to normal skin (NORM), psoriasis (PSOR) and five cases of ACD (CD1–5). ( c ) Nested PCR from same specimens was subjected to real-time PCR amplification, with SYBR green as the fluorochrome (* P
Figure Legend Snippet: Unique Vα24JαQ NKT-associated TCR gene expression occurs in ACD, but not in normal skin Nested PCR to detect unique Vα24-JαQ TCR sequences in skin biopsy specimens. RNA was extracted from an NKT cell line, Jurkat cells, and foreskin KC (FSKC), cDNA was synthesized, subjected to the nested PCR, and run on an agarose gel as described in Methods. ( a ) Control lane (Ctrl) represents no cDNA added to nested PCR. ( b ) This same nested PCR was applied to normal skin (NORM), psoriasis (PSOR) and five cases of ACD (CD1–5). ( c ) Nested PCR from same specimens was subjected to real-time PCR amplification, with SYBR green as the fluorochrome (* P

Techniques Used: Expressing, Nested PCR, Synthesized, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, SYBR Green Assay

CD1d+ Monocytic APCs, but not CD1d+ KC, induce cytokine gene expression and secretion by NKT-cells in vitro A polyclonal NKT cell line was cultured with the CD1d+ cell line, THP-1 or CD1d+ KC (differentiated or undifferentiated, and treated with IFN-γ, see Methods) in the presence ( a and b ) or absence (data not shown) of α-galactosylceramide for 24 hours, and supernatants were harvested for specific luminex assay. ( a ) IFN-γ; ( b ) IL-4. In another experiment, the NKT cells were isolated after 6 hours of incubation with the above cell lines, and RNA was extracted, cDNA was synthesized, and gene expression was studied by ( c ) real-time PCR IFN-γ; ( d ) IL-4. (* P
Figure Legend Snippet: CD1d+ Monocytic APCs, but not CD1d+ KC, induce cytokine gene expression and secretion by NKT-cells in vitro A polyclonal NKT cell line was cultured with the CD1d+ cell line, THP-1 or CD1d+ KC (differentiated or undifferentiated, and treated with IFN-γ, see Methods) in the presence ( a and b ) or absence (data not shown) of α-galactosylceramide for 24 hours, and supernatants were harvested for specific luminex assay. ( a ) IFN-γ; ( b ) IL-4. In another experiment, the NKT cells were isolated after 6 hours of incubation with the above cell lines, and RNA was extracted, cDNA was synthesized, and gene expression was studied by ( c ) real-time PCR IFN-γ; ( d ) IL-4. (* P

Techniques Used: Expressing, In Vitro, Cell Culture, Luminex, Isolation, Incubation, Synthesized, Real-time Polymerase Chain Reaction

9) Product Images from "Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells"

Article Title: Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084340

Comparison of Mcp-2 and Mcp-4 protein levels in mast cells from B6, B6-cma, and DBA/2 mice. Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. A. Cell extracts were resolved on 12.5% SDS gels followed by Coomassie blue staining (top panel) or western blotting with indicated antibodies. B. Cells were subjected to Wright-Giemsa staining (top panel) or immunofluorescent staining with indicated antibodies.
Figure Legend Snippet: Comparison of Mcp-2 and Mcp-4 protein levels in mast cells from B6, B6-cma, and DBA/2 mice. Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. A. Cell extracts were resolved on 12.5% SDS gels followed by Coomassie blue staining (top panel) or western blotting with indicated antibodies. B. Cells were subjected to Wright-Giemsa staining (top panel) or immunofluorescent staining with indicated antibodies.

Techniques Used: Mouse Assay, Derivative Assay, Staining, Western Blot

Identification of markedly increased expressions of Mcp-2 and Mcp-4 in BMMCs from a subpopulation of JAK2V617F transgenic mice. A. Detection of a predominant protein band in cell extracts of BMMCs from a variant line of mice. BMMCs from two JAK2V617F transgenic mice were extracted in a buffer containing 1% Triton X-100 or 1X SDS gel sample buffer were resolved on 10% SDS gel, and proteins were visualized by Coomassie blue staining. The arrow points to a predominant band. B. Verification of Mcp-2 and Mcp-4 over-expressions by Western blotting with specific antibodies. Extracts of BMMCs were separated on 12.5% SDS gel and subjected to Western blotting analyses with anti-Mcp-2 and Mcp-4. C. Verification of Mcp-2 and Mcp-4 over-expression by immunofluorescent cell staining. Mcp-2 and Mcp-4 were probed with specific antibodies followed by Cy-3-conjugated secondary antibodies (red). The nuclei (blue) were revealed by staining with Hoechst 33258.
Figure Legend Snippet: Identification of markedly increased expressions of Mcp-2 and Mcp-4 in BMMCs from a subpopulation of JAK2V617F transgenic mice. A. Detection of a predominant protein band in cell extracts of BMMCs from a variant line of mice. BMMCs from two JAK2V617F transgenic mice were extracted in a buffer containing 1% Triton X-100 or 1X SDS gel sample buffer were resolved on 10% SDS gel, and proteins were visualized by Coomassie blue staining. The arrow points to a predominant band. B. Verification of Mcp-2 and Mcp-4 over-expressions by Western blotting with specific antibodies. Extracts of BMMCs were separated on 12.5% SDS gel and subjected to Western blotting analyses with anti-Mcp-2 and Mcp-4. C. Verification of Mcp-2 and Mcp-4 over-expression by immunofluorescent cell staining. Mcp-2 and Mcp-4 were probed with specific antibodies followed by Cy-3-conjugated secondary antibodies (red). The nuclei (blue) were revealed by staining with Hoechst 33258.

Techniques Used: Transgenic Assay, Mouse Assay, Variant Assay, SDS-Gel, Staining, Western Blot, Over Expression

Identification of Mcp-2 and Mcp-4 gene variants. Schematic alignment of amino acid sequences (A.) and promoter region DNA sequences (B.) of Mcp-2P and Mcp-4P from control B6 and variant B6-cma mice. Variant amino acid residues and nucleotide bases are highlighted in bold. A vertical line “|” denotes identical amino acids or bases, and a dash “–” stands for deletions. Putative Mitf binding consensus motifs (CANNTG E-boxes) and an NdeI restriction cleavage site (CATATG) in the variant form of Mcp-2 or Mcp-4 are underlined. C. Detection of Mcp-2 and Mcp-4 gene variations in the promoter regions by allele-specific PCR and NdeI restriction fragment length polymorphism (RFLP), respectively. Normal form of Mcp-2P was detected by PCR with primers 5′-ctcacactggtcaacacaaacatta and 5′-tctgctgttaaacacaaacacagtct, while the Mcp-2P variant was amplified by using primers 5′-ctcacactggtcaacacaaacattg and 5′-tctgctgttaaacacaaacacagtca. The expected product size for both is 131 bp. The variant form of Mcp-4P was revealed by NdeI digestion which gave rise to two fragments while the normal form of Mcp-4P was not cleaved. Data show results for both homozygous and heterozygous mice.
Figure Legend Snippet: Identification of Mcp-2 and Mcp-4 gene variants. Schematic alignment of amino acid sequences (A.) and promoter region DNA sequences (B.) of Mcp-2P and Mcp-4P from control B6 and variant B6-cma mice. Variant amino acid residues and nucleotide bases are highlighted in bold. A vertical line “|” denotes identical amino acids or bases, and a dash “–” stands for deletions. Putative Mitf binding consensus motifs (CANNTG E-boxes) and an NdeI restriction cleavage site (CATATG) in the variant form of Mcp-2 or Mcp-4 are underlined. C. Detection of Mcp-2 and Mcp-4 gene variations in the promoter regions by allele-specific PCR and NdeI restriction fragment length polymorphism (RFLP), respectively. Normal form of Mcp-2P was detected by PCR with primers 5′-ctcacactggtcaacacaaacatta and 5′-tctgctgttaaacacaaacacagtct, while the Mcp-2P variant was amplified by using primers 5′-ctcacactggtcaacacaaacattg and 5′-tctgctgttaaacacaaacacagtca. The expected product size for both is 131 bp. The variant form of Mcp-4P was revealed by NdeI digestion which gave rise to two fragments while the normal form of Mcp-4P was not cleaved. Data show results for both homozygous and heterozygous mice.

Techniques Used: Variant Assay, Mouse Assay, Binding Assay, Polymerase Chain Reaction, Amplification

Association of Mcp-2 and Mcp-4 overexpressions in BMMCs with gene variants. A. Mice were genotyped for the Mcp-2 gene variant by allele-specific PCR and the Mcp-4 gene variant by restriction fragment length polymorphism (RFLP) with NdeI. BMMCs derived from these mice were analyzed for Mcp-2 and Mcp-4 protein expressions by Commassie blue staining and western blotting. B. BMMCs from B6 and B6-cma mice were analyzed for chymase activity. In at least 45 mice analyzed, there is a perfect correlation of Mcp-2 and Mcp-4 gene variants with overexpression of Mcp-2 and Mcp-4 and increased chymase activity.
Figure Legend Snippet: Association of Mcp-2 and Mcp-4 overexpressions in BMMCs with gene variants. A. Mice were genotyped for the Mcp-2 gene variant by allele-specific PCR and the Mcp-4 gene variant by restriction fragment length polymorphism (RFLP) with NdeI. BMMCs derived from these mice were analyzed for Mcp-2 and Mcp-4 protein expressions by Commassie blue staining and western blotting. B. BMMCs from B6 and B6-cma mice were analyzed for chymase activity. In at least 45 mice analyzed, there is a perfect correlation of Mcp-2 and Mcp-4 gene variants with overexpression of Mcp-2 and Mcp-4 and increased chymase activity.

Techniques Used: Mouse Assay, Variant Assay, Polymerase Chain Reaction, Derivative Assay, Staining, Western Blot, Activity Assay, Over Expression

10) Product Images from "Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells"

Article Title: Plant-derived SAC domain of PAR-4 (Prostate Apoptosis Response 4) exhibits growth inhibitory effects in prostate cancer cells

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2015.00822

Transgenic analyses. (A) PCR amplification of different genes, viz. SAC-Par-4 (i); rbcSE9 (ii) and npt II (iii) from transgenic lines VC, L2, L3, L5, and L6 on 1.5% agarose gel; (B) Southern blot analysis to investigate SAC-Par-4 gene insertion in transgenic plant lines VC, L2, L3, L5, and L6. Underneath is the agarose gel picture showing Xho I digested genomic DNA (10 μg) of T 2 generation of four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines (prior blotting); (C) Northern blot analysis of SAC-Par-4 mRNA expression in transgenic plants VC, L2, L3, L5, and L6. Underneath is an ethidium bromide-stained gel showing rRNA quality; (D) Real-time analysis of SAC-Par-4 transcripts in VC, L2, L3, L5, and L6 lines. The data were normalized by tubulin transcripts. The data shown are mean values from three independent experiments. Bars indicate the standard errors of means; (E) Nuclear run-on assay of SAC-Par-4 transcripts. Dot blots were hybridized to 32 P-labeled nascent transcripts from wild-type plant and transgenic lines L2, L3, and L5 which were synthesized by run-on transcription. pBSK plasmid (1 μg) and 18S ribosomal DNA (0.5 μg) were used as controls.
Figure Legend Snippet: Transgenic analyses. (A) PCR amplification of different genes, viz. SAC-Par-4 (i); rbcSE9 (ii) and npt II (iii) from transgenic lines VC, L2, L3, L5, and L6 on 1.5% agarose gel; (B) Southern blot analysis to investigate SAC-Par-4 gene insertion in transgenic plant lines VC, L2, L3, L5, and L6. Underneath is the agarose gel picture showing Xho I digested genomic DNA (10 μg) of T 2 generation of four transgenic (L2, L3, L5, and L6 of SAC-Par-4-GFP) and vector control lines (prior blotting); (C) Northern blot analysis of SAC-Par-4 mRNA expression in transgenic plants VC, L2, L3, L5, and L6. Underneath is an ethidium bromide-stained gel showing rRNA quality; (D) Real-time analysis of SAC-Par-4 transcripts in VC, L2, L3, L5, and L6 lines. The data were normalized by tubulin transcripts. The data shown are mean values from three independent experiments. Bars indicate the standard errors of means; (E) Nuclear run-on assay of SAC-Par-4 transcripts. Dot blots were hybridized to 32 P-labeled nascent transcripts from wild-type plant and transgenic lines L2, L3, and L5 which were synthesized by run-on transcription. pBSK plasmid (1 μg) and 18S ribosomal DNA (0.5 μg) were used as controls.

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation, Northern Blot, Expressing, Staining, Nuclear Run-on Assay, Labeling, Synthesized

Green Fluorescent Protein (GFP) analyses in transgenic tobacco lines. (A) PCR amplification of GFP (250 bp) using genomic DNA isolated from 10 independent transgenic plants (T 2 generation) expressing SAC-Par-4-GFP , vector control (VC) plant and wild-type plant; (B) The GFP protein concentration (expressed as μg GFP/mg protein) was measured in total soluble protein of transgenic and wild-type plants using Turner Biosystems Luminometer at GFP-UV module. Error bars indicate the standard deviation of readings from five different plants of each line (three readings per plant).
Figure Legend Snippet: Green Fluorescent Protein (GFP) analyses in transgenic tobacco lines. (A) PCR amplification of GFP (250 bp) using genomic DNA isolated from 10 independent transgenic plants (T 2 generation) expressing SAC-Par-4-GFP , vector control (VC) plant and wild-type plant; (B) The GFP protein concentration (expressed as μg GFP/mg protein) was measured in total soluble protein of transgenic and wild-type plants using Turner Biosystems Luminometer at GFP-UV module. Error bars indicate the standard deviation of readings from five different plants of each line (three readings per plant).

Techniques Used: Transgenic Assay, Polymerase Chain Reaction, Amplification, Isolation, Expressing, Plasmid Preparation, Protein Concentration, Standard Deviation

11) Product Images from "A novel testis-specific long noncoding RNA, Tesra, activates the Prss42/Tessp-2 gene during mouse spermatogenesis †"

Article Title: A novel testis-specific long noncoding RNA, Tesra, activates the Prss42/Tessp-2 gene during mouse spermatogenesis †

Journal: Biology of Reproduction

doi: 10.1093/biolre/ioy230

A hypothetical model for Prss42/Tessp-2 transcriptional activation. This figure indicates the hypothesized mechanism of Prss42/Tessp-2 gene activation based on our data. When spermatogonia divide into primary spermatocytes, the chromatin at the enhancer downstream of lncRNA-HSVIII begins to interact with that at the Prss42/Tessp-2 promoter, and thereby the enhancer is allowed to activate Prss42/Tessp-2 transcription. At a similar timing, Tesra transcripts are accumulated in the nuclei of primary spermatocytes and bind to chromatin at the Prss42/Tessp-2 promoter. The transcription level of Prss42/Tessp-2 is substantially enhanced by these two elements. There must be some proteins binding to Tesra transcripts and/or to the Prss42/Tessp-2 promoter.
Figure Legend Snippet: A hypothetical model for Prss42/Tessp-2 transcriptional activation. This figure indicates the hypothesized mechanism of Prss42/Tessp-2 gene activation based on our data. When spermatogonia divide into primary spermatocytes, the chromatin at the enhancer downstream of lncRNA-HSVIII begins to interact with that at the Prss42/Tessp-2 promoter, and thereby the enhancer is allowed to activate Prss42/Tessp-2 transcription. At a similar timing, Tesra transcripts are accumulated in the nuclei of primary spermatocytes and bind to chromatin at the Prss42/Tessp-2 promoter. The transcription level of Prss42/Tessp-2 is substantially enhanced by these two elements. There must be some proteins binding to Tesra transcripts and/or to the Prss42/Tessp-2 promoter.

Techniques Used: Activation Assay, Binding Assay

Tesra co-operatively increases Prss42/Tessp-2 promoter activity with the downstream enhancer. (A) A schematic view of the Prss/Tessp locus indicating positions of the enhancers. The Prss/Tessp locus is depicted as in Figure 1A . Two potential enhancers we identified before [ 45 ] are indicated with white boxes marked as “up” and “down.” (B) Successful induction of Tesra transcription in stable Hepa1–6 cells. We established stable Hepa1–6 cells that were responsive to Dox to induce Tesra transcription by the Tet-on system. Dox was added to the cells, and total RNAs were collected 24 h later. qRT-PCR was performed and the data are presented as in Figure 2D . Tesra transcription was significantly induced by the addition of Dox. The data are presented as means ± SD from three independent experiments and were analyzed by the Student t test. ** P
Figure Legend Snippet: Tesra co-operatively increases Prss42/Tessp-2 promoter activity with the downstream enhancer. (A) A schematic view of the Prss/Tessp locus indicating positions of the enhancers. The Prss/Tessp locus is depicted as in Figure 1A . Two potential enhancers we identified before [ 45 ] are indicated with white boxes marked as “up” and “down.” (B) Successful induction of Tesra transcription in stable Hepa1–6 cells. We established stable Hepa1–6 cells that were responsive to Dox to induce Tesra transcription by the Tet-on system. Dox was added to the cells, and total RNAs were collected 24 h later. qRT-PCR was performed and the data are presented as in Figure 2D . Tesra transcription was significantly induced by the addition of Dox. The data are presented as means ± SD from three independent experiments and were analyzed by the Student t test. ** P

Techniques Used: Activity Assay, Quantitative RT-PCR

Tesra occupancy at the Prss/Tessp locus. ChIRP-qPCR for Tesra in mouse testicular germ cells was performed. Germ cells were collected from 21- to 22-day-old mouse testes, and nuclear extracts were prepared. Sonicated chromatin was hybridized with biotinylated tiling oligo probes, and the bound chromatin was collected by streptavidin beads. Purified genome DNAs were investigated by qPCR. Rec8 and B2m promoters that were located on different chromosomes from the Prss/Tessp cluster were amplified as negative controls. The value was normalized to that of the input sample, which was kept before hybridization, and further normalized to the level at the Rec8 promoter (=1.0). The relative chromatin enrichment at each position is shown by the black bars. The white bars show the data from the experiment with RNase in hybridization buffer as a negative control. Positions of amplicons are indicated by arrows below the graph. The data are presented as means ± SD from three independent experiments and were analyzed by one-way ANOVA followed by the Tukey post hoc test. * P
Figure Legend Snippet: Tesra occupancy at the Prss/Tessp locus. ChIRP-qPCR for Tesra in mouse testicular germ cells was performed. Germ cells were collected from 21- to 22-day-old mouse testes, and nuclear extracts were prepared. Sonicated chromatin was hybridized with biotinylated tiling oligo probes, and the bound chromatin was collected by streptavidin beads. Purified genome DNAs were investigated by qPCR. Rec8 and B2m promoters that were located on different chromosomes from the Prss/Tessp cluster were amplified as negative controls. The value was normalized to that of the input sample, which was kept before hybridization, and further normalized to the level at the Rec8 promoter (=1.0). The relative chromatin enrichment at each position is shown by the black bars. The white bars show the data from the experiment with RNase in hybridization buffer as a negative control. Positions of amplicons are indicated by arrows below the graph. The data are presented as means ± SD from three independent experiments and were analyzed by one-way ANOVA followed by the Tukey post hoc test. * P

Techniques Used: Real-time Polymerase Chain Reaction, Sonication, Purification, Amplification, Hybridization, Negative Control

Cloning of a novel lncRNA, Tesra . (A) A schematic view of the Prss/Tessp locus on mouse chromosome 9. Exons of three Prss/Tessp genes are depicted by the black and white boxes representing translated and untranslated regions, respectively. Two lncRNAs that do not contain any introns are also depicted by the black boxes. The bent arrows indicate the transcriptional direction. Below the gene structure, the genomic region transcribed into Tesra is enlarged. A 2890-nucleotide sequence was first detected as a transcribed sequence by RT-PCR. 5′RACE was performed with three primers (GSP1–GSP3), and 3′RACE was performed with GSP4 and GSP5 primers. The resulting RACE products are indicated by horizontal lines. (B) 5′-end variations of Tesra determined by 5′RACE. A sequence around the 5′ end of Tesra that overlaps with the 3′ untranslated region of the Prss44/Tessp-4 gene is shown. The number of nucleotides between each sequence array is presented below. A specific band was obtained by 5′RACE, and 17 subclones were sequenced. The position of the 5′ end of each subclone is indicated by the red letters and the bent arrows with the number of subclones. Five TSSs were detected, and the most upstream adenine was determined to be a main TSS (indicated by a red arrow). (C) 3′ end of Tesra determined by 3′RACE. A specific band was obtained by 3′RACE, and 10 subclones were checked by DNA sequencing. In all subclones, an adenine was identified as a main transcriptional termination site, as indicated by a red letter and a vertical line.
Figure Legend Snippet: Cloning of a novel lncRNA, Tesra . (A) A schematic view of the Prss/Tessp locus on mouse chromosome 9. Exons of three Prss/Tessp genes are depicted by the black and white boxes representing translated and untranslated regions, respectively. Two lncRNAs that do not contain any introns are also depicted by the black boxes. The bent arrows indicate the transcriptional direction. Below the gene structure, the genomic region transcribed into Tesra is enlarged. A 2890-nucleotide sequence was first detected as a transcribed sequence by RT-PCR. 5′RACE was performed with three primers (GSP1–GSP3), and 3′RACE was performed with GSP4 and GSP5 primers. The resulting RACE products are indicated by horizontal lines. (B) 5′-end variations of Tesra determined by 5′RACE. A sequence around the 5′ end of Tesra that overlaps with the 3′ untranslated region of the Prss44/Tessp-4 gene is shown. The number of nucleotides between each sequence array is presented below. A specific band was obtained by 5′RACE, and 17 subclones were sequenced. The position of the 5′ end of each subclone is indicated by the red letters and the bent arrows with the number of subclones. Five TSSs were detected, and the most upstream adenine was determined to be a main TSS (indicated by a red arrow). (C) 3′ end of Tesra determined by 3′RACE. A specific band was obtained by 3′RACE, and 10 subclones were checked by DNA sequencing. In all subclones, an adenine was identified as a main transcriptional termination site, as indicated by a red letter and a vertical line.

Techniques Used: Clone Assay, Sequencing, Reverse Transcription Polymerase Chain Reaction, DNA Sequencing

Overexpression of Tesra increases Prss42/Tessp-2 expression and promoter activity. (A) Endogenous expression of Prss/Tessp cluster genes and Tesra in Hepa1–6 cells. qRT-PCR was performed with total RNAs from Hepa1–6 cells. Reverse transcription was done by using the oligo(dT) primer, and the Aip gene was examined as an internal control. Expression levels were normalized to Aip . (B) Successful overexpression of Tesra . Tesra-OE or the control vector was transiently transfected into Hepa1–6 cells, and after selection with G418, total RNA was purified from each sample. Complementary DNA was synthesized with the oligo(dT) primer, and PCR was conducted to detect Tesra and Gapdh expression. The cycle number is shown in parenthesis. A representative result from three experiments is shown. We could not see any difference among the three data sets. (C) Relative expression of Prss42/Tessp-2 mRNA by overexpression of Tesra . qRT-PCR was performed with the cDNAs prepared in (B). Data normalization was done by using Aip as an internal control. Transient overexpression of Tesra significantly increased Prss42/Tessp-2 expression in Hepa1–6 cells. The data are presented as means ± SD from three independent experiments and were analyzed by the Student t test. * P
Figure Legend Snippet: Overexpression of Tesra increases Prss42/Tessp-2 expression and promoter activity. (A) Endogenous expression of Prss/Tessp cluster genes and Tesra in Hepa1–6 cells. qRT-PCR was performed with total RNAs from Hepa1–6 cells. Reverse transcription was done by using the oligo(dT) primer, and the Aip gene was examined as an internal control. Expression levels were normalized to Aip . (B) Successful overexpression of Tesra . Tesra-OE or the control vector was transiently transfected into Hepa1–6 cells, and after selection with G418, total RNA was purified from each sample. Complementary DNA was synthesized with the oligo(dT) primer, and PCR was conducted to detect Tesra and Gapdh expression. The cycle number is shown in parenthesis. A representative result from three experiments is shown. We could not see any difference among the three data sets. (C) Relative expression of Prss42/Tessp-2 mRNA by overexpression of Tesra . qRT-PCR was performed with the cDNAs prepared in (B). Data normalization was done by using Aip as an internal control. Transient overexpression of Tesra significantly increased Prss42/Tessp-2 expression in Hepa1–6 cells. The data are presented as means ± SD from three independent experiments and were analyzed by the Student t test. * P

Techniques Used: Over Expression, Expressing, Activity Assay, Quantitative RT-PCR, Plasmid Preparation, Transfection, Selection, Purification, Synthesized, Polymerase Chain Reaction

12) Product Images from "Gene and protein expression of a soluble form of CTLA-4 in a healthy dog"

Article Title: Gene and protein expression of a soluble form of CTLA-4 in a healthy dog

Journal: The Journal of Veterinary Medical Science

doi: 10.1292/jvms.16-0583

RT-PCR amplification of canine CTLA-4 mRNA. Bands corresponding to 450 and 550 bp were observed. M; 100 bp DNA maker.
Figure Legend Snippet: RT-PCR amplification of canine CTLA-4 mRNA. Bands corresponding to 450 and 550 bp were observed. M; 100 bp DNA maker.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification

13) Product Images from "Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients"

Article Title: Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.9412

DNA sequencing from portions of the IL-4 gene from the forward strand are shown. The arrow indicates the position of SNP rs2070784 a: represents the genotype CC; b: TC and c: TT.
Figure Legend Snippet: DNA sequencing from portions of the IL-4 gene from the forward strand are shown. The arrow indicates the position of SNP rs2070784 a: represents the genotype CC; b: TC and c: TT.

Techniques Used: DNA Sequencing

Gel electrophoresis for (A) IL-4 PCR product visualized under UV light. M, 1000 bp marker, lane 1-10: from blood of glioma patients, lane 11-14: from blood of control group. The size of product is 280 bp and (B) IL-4R PCR product visualized under UV light. M, 1000 bp marker, lane 1-9: from blood of glioma patients, lane 10-11: from blood of control group. The size of product is 140 bp.
Figure Legend Snippet: Gel electrophoresis for (A) IL-4 PCR product visualized under UV light. M, 1000 bp marker, lane 1-10: from blood of glioma patients, lane 11-14: from blood of control group. The size of product is 280 bp and (B) IL-4R PCR product visualized under UV light. M, 1000 bp marker, lane 1-9: from blood of glioma patients, lane 10-11: from blood of control group. The size of product is 140 bp.

Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker

Mean serum concentration of IL-4 (pg/ml) in glioma patients and control group subjects as described in the materials and methods.
Figure Legend Snippet: Mean serum concentration of IL-4 (pg/ml) in glioma patients and control group subjects as described in the materials and methods.

Techniques Used: Concentration Assay

14) Product Images from "Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients"

Article Title: Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.9412

Gel electrophoresis for (A) IL-4 PCR product visualized under UV light. M, 1000 bp marker, lane 1-10: from blood of glioma patients, lane 11-14: from blood of control group. The size of product is 280 bp and (B) IL-4R PCR product visualized under UV light. M, 1000 bp marker, lane 1-9: from blood of glioma patients, lane 10-11: from blood of control group. The size of product is 140 bp.
Figure Legend Snippet: Gel electrophoresis for (A) IL-4 PCR product visualized under UV light. M, 1000 bp marker, lane 1-10: from blood of glioma patients, lane 11-14: from blood of control group. The size of product is 280 bp and (B) IL-4R PCR product visualized under UV light. M, 1000 bp marker, lane 1-9: from blood of glioma patients, lane 10-11: from blood of control group. The size of product is 140 bp.

Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker

15) Product Images from "Bone Marrow Stromal Cell Transdifferentiation into Oligodendrocyte-Like Cells Using Triiodothyronine as a Inducer with Expression of Platelet-Derived Growth Factor ? as a Maturity Marker"

Article Title: Bone Marrow Stromal Cell Transdifferentiation into Oligodendrocyte-Like Cells Using Triiodothyronine as a Inducer with Expression of Platelet-Derived Growth Factor ? as a Maturity Marker

Journal: Iranian Biomedical Journal

doi: 10.6091/ibj.11162.2013

Electrophorograms of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH , 400 bp),  NeuroD  (220 bp),  Oct -4  (210 bp) and platelet-derived growth factor α ( PDGFR-α , 190 bp) using RT-PCR.  (A)  The electrophorogram of  NeuroD  gene expression profile
Figure Legend Snippet: Electrophorograms of glyceraldehyde 3-phosphate dehydrogenase ( GAPDH , 400 bp), NeuroD (220 bp), Oct -4 (210 bp) and platelet-derived growth factor α ( PDGFR-α , 190 bp) using RT-PCR. (A) The electrophorogram of NeuroD gene expression profile

Techniques Used: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

16) Product Images from "Identification of a Secreted Casein Kinase 1 in Leishmania donovani: Effect of Protein over Expression on Parasite Growth and Virulence"

Article Title: Identification of a Secreted Casein Kinase 1 in Leishmania donovani: Effect of Protein over Expression on Parasite Growth and Virulence

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079287

Comparison of wild-type and mutant promastigote growth and metacyclogenesis in culture. Promastigotes, Ld:wild type (wt), Ld:LUC (luc) and Ld:CK1.4-FLAG (CK1.4) expressing mutants, were diluted (10 5 cells/ml) at day 0 in complete culture media. Panel A. Parasite growth was monitored daily by counting live cells in a haemocytometer. All experiments were performed in triplicates. *, P
Figure Legend Snippet: Comparison of wild-type and mutant promastigote growth and metacyclogenesis in culture. Promastigotes, Ld:wild type (wt), Ld:LUC (luc) and Ld:CK1.4-FLAG (CK1.4) expressing mutants, were diluted (10 5 cells/ml) at day 0 in complete culture media. Panel A. Parasite growth was monitored daily by counting live cells in a haemocytometer. All experiments were performed in triplicates. *, P

Techniques Used: Mutagenesis, Expressing

Localization of CK1.4 in Leishmania donovani promastigotes by immunefluorescent staining with anti-FLAG and anti-CK1.4 antibodies. Logarithmic stage Ld:CK1.4-FLAG (Panels Ld:CK1.4) and Ld:LUC (Panels Ld:luc) promastigotes were fixed in 4% paraformaldehyde, centrifuged onto poly-L-lysine coated slides, and permeabilized with ice cold methanol. The slides were incubated with anti-FLAG M2 monoclonal antibodies (1/500 dilution), rabbit anti-CK1.4 polyclonal antibodies (1/7500 dilution), or buffer alone. Staining was carried out by incubating slides in appropriate secondary antibody, either Cy2-goat anti-mouse IgG or Cy3-goat anti-rabbit IgG (Panel Cy2/3; 1/200 dilution), and mounted in Fluoroshield with DAPI (Panel DAPI). The immunofluorescence examined using an Apochromat oil immersion objective (100×magnification) on an Olympus IX71S8F microscope.
Figure Legend Snippet: Localization of CK1.4 in Leishmania donovani promastigotes by immunefluorescent staining with anti-FLAG and anti-CK1.4 antibodies. Logarithmic stage Ld:CK1.4-FLAG (Panels Ld:CK1.4) and Ld:LUC (Panels Ld:luc) promastigotes were fixed in 4% paraformaldehyde, centrifuged onto poly-L-lysine coated slides, and permeabilized with ice cold methanol. The slides were incubated with anti-FLAG M2 monoclonal antibodies (1/500 dilution), rabbit anti-CK1.4 polyclonal antibodies (1/7500 dilution), or buffer alone. Staining was carried out by incubating slides in appropriate secondary antibody, either Cy2-goat anti-mouse IgG or Cy3-goat anti-rabbit IgG (Panel Cy2/3; 1/200 dilution), and mounted in Fluoroshield with DAPI (Panel DAPI). The immunofluorescence examined using an Apochromat oil immersion objective (100×magnification) on an Olympus IX71S8F microscope.

Techniques Used: Staining, Incubation, Immunofluorescence, Microscopy

17) Product Images from "Functional Desaturase Fads1 (Δ5) and Fads2 (Δ6) Orthologues Evolved before the Origin of Jawed Vertebrates"

Article Title: Functional Desaturase Fads1 (Δ5) and Fads2 (Δ6) Orthologues Evolved before the Origin of Jawed Vertebrates

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031950

Functional characterization of the newly cloned Scyliorhinus canicula fatty acyl desaturases FADS1. (Panels A, C and E) and FADS2 (panels B, D and F) in transgenic yeast ( Saccharomyces cerevisiae ) grown in the presence of Δ6 substrates 18:3n-3 (A and B), Δ5 substrates 20:4n-3 (C and D) and Δ4 substrates (E and F). Fatty acids were extracted from yeast transformed with pYES2 vector containing the ORF of the putative fatty acyl desaturase cDNA as an insert. The first four peaks in all panels are the main endogenous fatty acids of S. cerevisiae , namely 16:0 (1), 16:1 isomers (2), 18:0 (3), and 18:1n-9 (4). Substrates (“*”) and their corresponding desaturated products are indicated accordingly in panels A–F. Vertical axis, FID response; horizontal axis, retention time.
Figure Legend Snippet: Functional characterization of the newly cloned Scyliorhinus canicula fatty acyl desaturases FADS1. (Panels A, C and E) and FADS2 (panels B, D and F) in transgenic yeast ( Saccharomyces cerevisiae ) grown in the presence of Δ6 substrates 18:3n-3 (A and B), Δ5 substrates 20:4n-3 (C and D) and Δ4 substrates (E and F). Fatty acids were extracted from yeast transformed with pYES2 vector containing the ORF of the putative fatty acyl desaturase cDNA as an insert. The first four peaks in all panels are the main endogenous fatty acids of S. cerevisiae , namely 16:0 (1), 16:1 isomers (2), 18:0 (3), and 18:1n-9 (4). Substrates (“*”) and their corresponding desaturated products are indicated accordingly in panels A–F. Vertical axis, FID response; horizontal axis, retention time.

Techniques Used: Functional Assay, Clone Assay, Transgenic Assay, Transformation Assay, Plasmid Preparation

18) Product Images from "Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells"

Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085740

Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.
Figure Legend Snippet: Detection of the HS II site of the IL-4 gene in Th2 and Th1 cells. A. Detection of HS II site using DHS libraries as templates. Upper panel shows the positions of the HS II site and the PCR primers at the IL-4 gene locus. The lower panel shows real-time PCR results using DHS libraries as templates and the indicated primer pairs. B. Detection of the HS II site using unpurified DNA as templates. Adaptor-ligated high-molecular-weight DNA derived from nuclei of Th2 and Th1 cells with or without DNase I digestion were used as templates in real-time PCR. Results with the indicated primer pair are shown. In all panels of the figure, relative amplification signals were determined by comparing to that of DNase I-undigested samples of the respective cell type.

Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Molecular Weight, Derivative Assay, Amplification

19) Product Images from "Early detection of Y chromosome microdeletions in infertile men is helpful to guide clinical reproductive treatments in southwest of China"

Article Title: Early detection of Y chromosome microdeletions in infertile men is helpful to guide clinical reproductive treatments in southwest of China

Journal: Medicine

doi: 10.1097/MD.0000000000014350

Y chromosome microdeletions detected by multiplex PCR using AZF specific STSs makers. (A) Multiplex PCR amplification results for various Y chromosome microdeletion types in multiplex A: SYR (FAM), sY84 (VIC), sY127 (ROX), sY255 (Cy5) and multiplex B: ZFX/ZFY (FAM), sY86 (VIC), sY134 (ROX), sY254 (Cy5). AZFa (sY84, sY86), AZFb (sY127, sY134), AZFc (sY254, sY255), AZFb + c and AZFa + b + c deletions were detected in different patients. gDNA from healthy male were used as the positive control. gDNA from female and water was used as the negative control. SYR and ZFX/ZFY were used as the internal reference. AZF = azoospermia factor, gDNA = genomic DNA, PCR = polymerase chain reaction, SYR = sex-determining region of the Y chromosome, ZFX = zinc finger protein, X-linked, ZFY = zinc finger protein, Y-linked.
Figure Legend Snippet: Y chromosome microdeletions detected by multiplex PCR using AZF specific STSs makers. (A) Multiplex PCR amplification results for various Y chromosome microdeletion types in multiplex A: SYR (FAM), sY84 (VIC), sY127 (ROX), sY255 (Cy5) and multiplex B: ZFX/ZFY (FAM), sY86 (VIC), sY134 (ROX), sY254 (Cy5). AZFa (sY84, sY86), AZFb (sY127, sY134), AZFc (sY254, sY255), AZFb + c and AZFa + b + c deletions were detected in different patients. gDNA from healthy male were used as the positive control. gDNA from female and water was used as the negative control. SYR and ZFX/ZFY were used as the internal reference. AZF = azoospermia factor, gDNA = genomic DNA, PCR = polymerase chain reaction, SYR = sex-determining region of the Y chromosome, ZFX = zinc finger protein, X-linked, ZFY = zinc finger protein, Y-linked.

Techniques Used: Multiplex Assay, Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

20) Product Images from "Pol? ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability"

Article Title: Pol? ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20080669

GC formation in Polζ f/Δ /CD21-cre mice. (A) Generation of GC B cells. Mice were immunized with 100 µg NP-CG in alum and analyzed for the presence of CD19 + PNA + Fas + GC B cells 14 d after immunization. Only CD19 + cells are shown. SPL, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes. Data represent one of two independent experiments. (B) Deletion efficiency of the Polζ f allele. The Polζ f and Polζ Δ alleles in B cells of Polζ f/Δ /CD21-cre and control mice were amplified in a competitive PCR using primers that anneal 5′, within, and 3′ of the floxed region and compared with a standard with known ratios of the two alleles. The fragments corresponding to the floxed and deleted allele migrate at around 300 and 450 bp, respectively.
Figure Legend Snippet: GC formation in Polζ f/Δ /CD21-cre mice. (A) Generation of GC B cells. Mice were immunized with 100 µg NP-CG in alum and analyzed for the presence of CD19 + PNA + Fas + GC B cells 14 d after immunization. Only CD19 + cells are shown. SPL, spleen; PP, Peyer's patches; MLN, mesenteric lymph nodes. Data represent one of two independent experiments. (B) Deletion efficiency of the Polζ f allele. The Polζ f and Polζ Δ alleles in B cells of Polζ f/Δ /CD21-cre and control mice were amplified in a competitive PCR using primers that anneal 5′, within, and 3′ of the floxed region and compared with a standard with known ratios of the two alleles. The fragments corresponding to the floxed and deleted allele migrate at around 300 and 450 bp, respectively.

Techniques Used: Mouse Assay, Amplification, Polymerase Chain Reaction

Single cell analysis of SHM in Polζ f/Δ /CD21-cre mice. (A) Distribution of mutations per B cell and mutation frequency in Polζ -deficient and Polζ -proficient B cells. Single GC B cells were sorted by FACS 14 d after immunization with NP-CG. PCR fragments containing the introns downstream of the rearranged V(D)J elements were amplified in a seminested PCR and subsequently sequenced. Simultaneously, the single cells were genotyped for the presence of the Polζ f allele. The mutation frequency was determined by the number of mutations present in the sequence of 500 bp, downstream of the individual VDJ rearrangement. (B) To compare the patterns of mutations of Polζ-deficient and -proficient cells, sequences derived from B cells using the same rearranged J H element were grouped together and the mutations in each group were counted. The emerging mutation pattern in each group was corrected for the base composition of that particular intron before the patterns of the four J H introns were compiled together. All values are shown in percentages and were rounded to the nearest whole number. n = the number of mutations; Tr./Tv., the transitions (Tr.) over transversions (Tv.) ratio. Shown are the combined mutation patterns corrected for the base composition of the sequences. (C) Percentage of mutations at A-T versus C-G basepairs in the same analysis.
Figure Legend Snippet: Single cell analysis of SHM in Polζ f/Δ /CD21-cre mice. (A) Distribution of mutations per B cell and mutation frequency in Polζ -deficient and Polζ -proficient B cells. Single GC B cells were sorted by FACS 14 d after immunization with NP-CG. PCR fragments containing the introns downstream of the rearranged V(D)J elements were amplified in a seminested PCR and subsequently sequenced. Simultaneously, the single cells were genotyped for the presence of the Polζ f allele. The mutation frequency was determined by the number of mutations present in the sequence of 500 bp, downstream of the individual VDJ rearrangement. (B) To compare the patterns of mutations of Polζ-deficient and -proficient cells, sequences derived from B cells using the same rearranged J H element were grouped together and the mutations in each group were counted. The emerging mutation pattern in each group was corrected for the base composition of that particular intron before the patterns of the four J H introns were compiled together. All values are shown in percentages and were rounded to the nearest whole number. n = the number of mutations; Tr./Tv., the transitions (Tr.) over transversions (Tv.) ratio. Shown are the combined mutation patterns corrected for the base composition of the sequences. (C) Percentage of mutations at A-T versus C-G basepairs in the same analysis.

Techniques Used: Single-cell Analysis, Mouse Assay, Mutagenesis, FACS, Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay

21) Product Images from "Claudin-4 knockout by TALEN-mediated gene targeting in MDCK cells: Claudin-4 is dispensable for the permeability properties of tight junctions in wild-type MDCK cells"

Article Title: Claudin-4 knockout by TALEN-mediated gene targeting in MDCK cells: Claudin-4 is dispensable for the permeability properties of tight junctions in wild-type MDCK cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0182521

Establishment of claudin-4 knockout clones in MDCK II cells. (A) Immunofluorescence analysis of claudin-4 and occludin in wild-type cells (CTL) and claudin-4 knockout clones (sKO1–3) in MDCK II cells. Claudin-4 staining at cell-cell contacts was completely lost in claudin-4 knockout clones. Scale bar = 10 μm. (B) Immunoblots of claudin-4 and E-cadherin in wild-type cells and claudin-2 knockout clones. A claudin-4 band of ~20 kDa was absent in claudin-4 knockout clones. (C) DNA sequences of the TALEN targeting site in wild-type cells and claudin-4 knockout clones. Dash indicates loss of a nucleotide and green letters indicate additional nucleotides. Frame shifts were confirmed in all clones. (D) Genomic PCR analysis of wild-type cells and claudin-4 knockout clones using primers for TALEN and claudin-4 DNAs. A clone stably expressing TALEN was used as a positive control (PC). None of the PCR products for TALENs was detected in claudin-4 knockout clones.
Figure Legend Snippet: Establishment of claudin-4 knockout clones in MDCK II cells. (A) Immunofluorescence analysis of claudin-4 and occludin in wild-type cells (CTL) and claudin-4 knockout clones (sKO1–3) in MDCK II cells. Claudin-4 staining at cell-cell contacts was completely lost in claudin-4 knockout clones. Scale bar = 10 μm. (B) Immunoblots of claudin-4 and E-cadherin in wild-type cells and claudin-2 knockout clones. A claudin-4 band of ~20 kDa was absent in claudin-4 knockout clones. (C) DNA sequences of the TALEN targeting site in wild-type cells and claudin-4 knockout clones. Dash indicates loss of a nucleotide and green letters indicate additional nucleotides. Frame shifts were confirmed in all clones. (D) Genomic PCR analysis of wild-type cells and claudin-4 knockout clones using primers for TALEN and claudin-4 DNAs. A clone stably expressing TALEN was used as a positive control (PC). None of the PCR products for TALENs was detected in claudin-4 knockout clones.

Techniques Used: Knock-Out, Clone Assay, Immunofluorescence, CTL Assay, Staining, Western Blot, Polymerase Chain Reaction, Stable Transfection, Expressing, Positive Control, TALENs

Establishment of claudin-2 and claudin-4 double knockout clones in MDCK II cells. (A) Genomic PCR analysis of wild-type cells, claudin-4 knockout clones (sKO4, sKO5) and claudin-2 and claudin-4 double knockout clones (dKO4–6) using primers for the TALEN targeting site in claudin-4 genes. (B) DNA sequences of the TALEN targeting site in wild-type cells and dKO4 and dKO6 clones. Green letters indicate additional nucleotides. (C) Genomic PCR analysis of wild-type cells and sKO4, sKO5 and dKO5 clones using primers for the region of sequences containing 400 bases before the TALEN targeting site. (D) Genomic PCR analysis of wild-type cells and sKO4, sKO5 and dKO5 clones using primers for the region within the claudin-4 gene. (E) Immunoblots of claudin-1, -2, -3, -4 and -7 in claudin-4 knockout clones.
Figure Legend Snippet: Establishment of claudin-2 and claudin-4 double knockout clones in MDCK II cells. (A) Genomic PCR analysis of wild-type cells, claudin-4 knockout clones (sKO4, sKO5) and claudin-2 and claudin-4 double knockout clones (dKO4–6) using primers for the TALEN targeting site in claudin-4 genes. (B) DNA sequences of the TALEN targeting site in wild-type cells and dKO4 and dKO6 clones. Green letters indicate additional nucleotides. (C) Genomic PCR analysis of wild-type cells and sKO4, sKO5 and dKO5 clones using primers for the region of sequences containing 400 bases before the TALEN targeting site. (D) Genomic PCR analysis of wild-type cells and sKO4, sKO5 and dKO5 clones using primers for the region within the claudin-4 gene. (E) Immunoblots of claudin-1, -2, -3, -4 and -7 in claudin-4 knockout clones.

Techniques Used: Double Knockout, Clone Assay, Polymerase Chain Reaction, Knock-Out, Western Blot

Establishment of claudin-2 and claudin-4 double knockout clones in MDCK II cells. (A) Immunofluorescence analysis of claudin-4 and occludin in claudin-2 knockout clone (CTL) and claudin-2 and claudin-4 double knockout clones (dKO1–3) in MDCK II cells. Claudin-4 staining at cell-cell contacts was completely lost in double knockout clones. Scale bar = 10 μm. (B) Immunoblots of claudin-4 and E-cadherin in claudin-2 knockout clone and double knockout clones. (C) DNA sequences of the TALEN targeting site in wild-type cells and double knockout clones. One type of mutation was found in the alleles of the dKO3 clone and two types in the alleles of the dKO1 and dKO2 clones. Green letters indicate additional nucleotides. Frame shifts were confirmed in all alleles. (D) Genomic PCR analysis of wild-type cells and double knockout clones using primers for TALEN and claudin-4 DNAs. None of the PCR products for TALENs was detected in double knockout clones.
Figure Legend Snippet: Establishment of claudin-2 and claudin-4 double knockout clones in MDCK II cells. (A) Immunofluorescence analysis of claudin-4 and occludin in claudin-2 knockout clone (CTL) and claudin-2 and claudin-4 double knockout clones (dKO1–3) in MDCK II cells. Claudin-4 staining at cell-cell contacts was completely lost in double knockout clones. Scale bar = 10 μm. (B) Immunoblots of claudin-4 and E-cadherin in claudin-2 knockout clone and double knockout clones. (C) DNA sequences of the TALEN targeting site in wild-type cells and double knockout clones. One type of mutation was found in the alleles of the dKO3 clone and two types in the alleles of the dKO1 and dKO2 clones. Green letters indicate additional nucleotides. Frame shifts were confirmed in all alleles. (D) Genomic PCR analysis of wild-type cells and double knockout clones using primers for TALEN and claudin-4 DNAs. None of the PCR products for TALENs was detected in double knockout clones.

Techniques Used: Double Knockout, Clone Assay, Immunofluorescence, Knock-Out, CTL Assay, Staining, Western Blot, Mutagenesis, Polymerase Chain Reaction, TALENs

22) Product Images from "Preliminary analysis of single-nucleotide polymorphisms in IL-10, IL-4, and IL-4Rα genes and profile of circulating cytokines in patients with gastric Cancer"

Article Title: Preliminary analysis of single-nucleotide polymorphisms in IL-10, IL-4, and IL-4Rα genes and profile of circulating cytokines in patients with gastric Cancer

Journal: BMC Gastroenterology

doi: 10.1186/s12876-018-0913-9

Level of peripheral blood circulating cytokines. The amount of cytokines (pg/mL) is shown: a IL-4; ( b ). TNF-α; ( c ). IL-10, and ( b ). IFN-γ in serum from patients with gastric carcinoma, GC (pink filled circles represent each individual) and in healthy population (green empty diamonds per individual). The line corresponds to the median value. *( P
Figure Legend Snippet: Level of peripheral blood circulating cytokines. The amount of cytokines (pg/mL) is shown: a IL-4; ( b ). TNF-α; ( c ). IL-10, and ( b ). IFN-γ in serum from patients with gastric carcinoma, GC (pink filled circles represent each individual) and in healthy population (green empty diamonds per individual). The line corresponds to the median value. *( P

Techniques Used:

23) Product Images from "Staphylococcus aureus enterotoxins modulate IL-22-secreting cells in adults with atopic dermatitis"

Article Title: Staphylococcus aureus enterotoxins modulate IL-22-secreting cells in adults with atopic dermatitis

Journal: Scientific Reports

doi: 10.1038/s41598-018-25125-0

Cytokine gene expression profiles in AD skin. Expression profiles of IL-22, IL-4, IL-10, and TNF-α in skin of the healthy control group (HC, n = 5) compared with AD patients (AD, n = 10), assessed by real-time PCR. Lines represent medians with interquartile ranges of cytokines in skin specimens. *p ≤ 0.05 and **p ≤ 0.01.
Figure Legend Snippet: Cytokine gene expression profiles in AD skin. Expression profiles of IL-22, IL-4, IL-10, and TNF-α in skin of the healthy control group (HC, n = 5) compared with AD patients (AD, n = 10), assessed by real-time PCR. Lines represent medians with interquartile ranges of cytokines in skin specimens. *p ≤ 0.05 and **p ≤ 0.01.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

24) Product Images from "The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription"

Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

Journal: Virology Journal

doi: 10.1186/s12985-016-0600-9

The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P
Figure Legend Snippet: The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Injection

UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P
Figure Legend Snippet: UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P

Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase

Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box
Figure Legend Snippet: Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box

Techniques Used: Mutagenesis, Labeling, Amplification, Expressing, Infection, Polymerase Chain Reaction

The mutated UL7 gene modulates viral proliferation. a Dynamic profile of the UL7-MU viral strain at 37 °C. Vero cells were infected with UL7-MU (filled circles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells. b Morphological features of UL7-MU mutant plaques at 37 °C. Vero cells were infected with UL7-MU or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profile of the UL7-MU viral strain at 34 °C. Vero cells were infected with UL7-MU (filled triangles) or HSV-1 WT (open triangles) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells
Figure Legend Snippet: The mutated UL7 gene modulates viral proliferation. a Dynamic profile of the UL7-MU viral strain at 37 °C. Vero cells were infected with UL7-MU (filled circles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells. b Morphological features of UL7-MU mutant plaques at 37 °C. Vero cells were infected with UL7-MU or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profile of the UL7-MU viral strain at 34 °C. Vero cells were infected with UL7-MU (filled triangles) or HSV-1 WT (open triangles) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells

Techniques Used: Infection, Mutagenesis, Staining

The UL7-MU viral strain exhibits lower virulence in infected mice compared with the WT strain. a BALB/c mice were infected via intranasal instillation of 2.3 × 10 5 PFU of WT HSV-1 (open boxes), UL7-MU (filled circles), MU-complemented P1 (filled triangles) or PBS (open rhombuses) as a control. The weights of the mice were measured every 2 days. Data are shown as means ± SD. ∗ P
Figure Legend Snippet: The UL7-MU viral strain exhibits lower virulence in infected mice compared with the WT strain. a BALB/c mice were infected via intranasal instillation of 2.3 × 10 5 PFU of WT HSV-1 (open boxes), UL7-MU (filled circles), MU-complemented P1 (filled triangles) or PBS (open rhombuses) as a control. The weights of the mice were measured every 2 days. Data are shown as means ± SD. ∗ P

Techniques Used: Infection, Mouse Assay

The mutated phenotype of UL7-MU is complemented by expression of the UL7 gene in cells. a The lysates of Vero cells transfected with the pcDNA3-UL7 plasmid were separated on a 12 % polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with UL7 antibody. The position of the UL7 protein is indicated. b Morphological features of plaques formed by the complemented UL7-MU and WT viral strains. Cells were infected at 24 h post-transfection with UL7-MU or WT virus at an MOI of 1. The supernatants and cells of the complemented virus were harvested after CPE was observed, followed by plaque assays. Vero cells were infected with MU-complemented P1, WT-control P1, MU-complemented P2, WT-control P2 or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profiles of the MU-complemented P1 and MU-complemented P2 viral strains at 37 °C. Vero cells were infected with the MU-complemented P1 (filled circles), MU-complemented P2 (filled triangles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells
Figure Legend Snippet: The mutated phenotype of UL7-MU is complemented by expression of the UL7 gene in cells. a The lysates of Vero cells transfected with the pcDNA3-UL7 plasmid were separated on a 12 % polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with UL7 antibody. The position of the UL7 protein is indicated. b Morphological features of plaques formed by the complemented UL7-MU and WT viral strains. Cells were infected at 24 h post-transfection with UL7-MU or WT virus at an MOI of 1. The supernatants and cells of the complemented virus were harvested after CPE was observed, followed by plaque assays. Vero cells were infected with MU-complemented P1, WT-control P1, MU-complemented P2, WT-control P2 or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profiles of the MU-complemented P1 and MU-complemented P2 viral strains at 37 °C. Vero cells were infected with the MU-complemented P1 (filled circles), MU-complemented P2 (filled triangles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells

Techniques Used: Expressing, Transfection, Plasmid Preparation, Infection, Staining

25) Product Images from "Banana-Associated Microbial Communities in Uganda Are Highly Diverse but Dominated by Enterobacteriaceae"

Article Title: Banana-Associated Microbial Communities in Uganda Are Highly Diverse but Dominated by Enterobacteriaceae

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00772-12

Molecular fingerprints of 16S rRNA gene fragments amplified from endosphere samples of all four field sites and separated by SSCP. Four lanes of each variant represent independent replicates. Lanes S, 1-kb ladder. Marked bands were sequenced and compared to those in the GenBank database using the BLAST algorithm. Detailed results are presented in Table S1 in the supplemental material.
Figure Legend Snippet: Molecular fingerprints of 16S rRNA gene fragments amplified from endosphere samples of all four field sites and separated by SSCP. Four lanes of each variant represent independent replicates. Lanes S, 1-kb ladder. Marked bands were sequenced and compared to those in the GenBank database using the BLAST algorithm. Detailed results are presented in Table S1 in the supplemental material.

Techniques Used: Amplification, Variant Assay

BOX patterns of bacterial antagonists of Burkholderia species and Serratia marcescens isolates obtained from the rhizosphere (R), endosphere (E), and soil (S) of the investigated field sites.
Figure Legend Snippet: BOX patterns of bacterial antagonists of Burkholderia species and Serratia marcescens isolates obtained from the rhizosphere (R), endosphere (E), and soil (S) of the investigated field sites.

Techniques Used:

16S rRNA copy numbers per gram of fresh weight (g fw −1 ) determined by Enterobacteriaceae -specific quantitative PCR. Log 10 values are shown for the rhizosphere (R), the endosphere (E), and soil (S) for the four investigated field sites: field 1, no herbicide treatment, no agro-forest; field 2, intensive herbicide treatment, no agro-forest; field 3a, no herbicide treatment, agro-forest; and field 3b, no herbicide treatment, no agro-forest. Error bars indicate confidence intervals at P = 0.005.
Figure Legend Snippet: 16S rRNA copy numbers per gram of fresh weight (g fw −1 ) determined by Enterobacteriaceae -specific quantitative PCR. Log 10 values are shown for the rhizosphere (R), the endosphere (E), and soil (S) for the four investigated field sites: field 1, no herbicide treatment, no agro-forest; field 2, intensive herbicide treatment, no agro-forest; field 3a, no herbicide treatment, agro-forest; and field 3b, no herbicide treatment, no agro-forest. Error bars indicate confidence intervals at P = 0.005.

Techniques Used: Real-time Polymerase Chain Reaction

Occurrence of Enterobacteriaceae in the endosphere and endorhiza of banana plants. FISH with the probes EnterbactD-ATTO488, ALF968-Cy5, and EUBMIX-Cy3 of banana thin sections and CLSM showed colonization of the endosphere (A, B, and C) and endorhiza (D). (A and B) Violet, Enterobacteriaceae ; yellow, Alphaproteobacteria ; red, other bacteria. Panel B shows the thre-dimensional computer reconstruction of panel A, using Imaris7.0. (C) Violet, Enterobacteriaceae (arrows); yellow, Alphaproteobacteria ; red, other bacteria or plant structures (long, parallel bars). (D) Violet, Enterobacteriaceae (arrows) or plant cell walls; yellow, Alphaproteobacteria ; red: other bacteria. Scale bars, 20 μm.
Figure Legend Snippet: Occurrence of Enterobacteriaceae in the endosphere and endorhiza of banana plants. FISH with the probes EnterbactD-ATTO488, ALF968-Cy5, and EUBMIX-Cy3 of banana thin sections and CLSM showed colonization of the endosphere (A, B, and C) and endorhiza (D). (A and B) Violet, Enterobacteriaceae ; yellow, Alphaproteobacteria ; red, other bacteria. Panel B shows the thre-dimensional computer reconstruction of panel A, using Imaris7.0. (C) Violet, Enterobacteriaceae (arrows); yellow, Alphaproteobacteria ; red, other bacteria or plant structures (long, parallel bars). (D) Violet, Enterobacteriaceae (arrows) or plant cell walls; yellow, Alphaproteobacteria ; red: other bacteria. Scale bars, 20 μm.

Techniques Used: Fluorescence In Situ Hybridization, Confocal Laser Scanning Microscopy

26) Product Images from "Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin"

Article Title: Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin

Journal: Research in Pharmaceutical Sciences

doi: 10.4103/1735-5362.235162

Chromosomal DNA amplification. Agarose gel electrophoresis results illustrated the insertion of truncated α4 integrin ( TITGA4) into the genome of transfected cells. Lane 1-3, amplified 879 bp fragment of transfected cells; lane 4, untransfected cell, and lane 5, marker 1 kb.
Figure Legend Snippet: Chromosomal DNA amplification. Agarose gel electrophoresis results illustrated the insertion of truncated α4 integrin ( TITGA4) into the genome of transfected cells. Lane 1-3, amplified 879 bp fragment of transfected cells; lane 4, untransfected cell, and lane 5, marker 1 kb.

Techniques Used: Amplification, Agarose Gel Electrophoresis, Transfection, Marker

27) Product Images from "Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin"

Article Title: Construction and characterization of human embryonic kidney-(HEK)-293T cell overexpressing truncated α4 integrin

Journal: Research in Pharmaceutical Sciences

doi: 10.4103/1735-5362.235162

Chromosomal DNA amplification. Agarose gel electrophoresis results illustrated the insertion of truncated α4 integrin ( TITGA4) into the genome of transfected cells. Lane 1-3, amplified 879 bp fragment of transfected cells; lane 4, untransfected cell, and lane 5, marker 1 kb.
Figure Legend Snippet: Chromosomal DNA amplification. Agarose gel electrophoresis results illustrated the insertion of truncated α4 integrin ( TITGA4) into the genome of transfected cells. Lane 1-3, amplified 879 bp fragment of transfected cells; lane 4, untransfected cell, and lane 5, marker 1 kb.

Techniques Used: Amplification, Agarose Gel Electrophoresis, Transfection, Marker

28) Product Images from "Experimental Lyme Arthritis in the Absence of Interleukin-4 or Gamma Interferon"

Article Title: Experimental Lyme Arthritis in the Absence of Interleukin-4 or Gamma Interferon

Journal: Infection and Immunity

doi:

Competitive PCR amplification of ospA and IL-4pr genes from ankles of C3H, C3H IFN-γ°, DBA, and DBA IL-4° mice. The control lane contains DNA from an uninfected mouse. The upper bands in each lane are the BC3 competitor amplification products, and the lower bands are wild-type DNA PCR products. Each lane represents data from an individual mouse. Sample DNA levels were equalized by using primers for the single-copy mammalian IL-4pr gene. Spirochete DNA was amplified by using primers for ospA . The amount of BC3 competitor spiked into each sample was 0.25 pg.
Figure Legend Snippet: Competitive PCR amplification of ospA and IL-4pr genes from ankles of C3H, C3H IFN-γ°, DBA, and DBA IL-4° mice. The control lane contains DNA from an uninfected mouse. The upper bands in each lane are the BC3 competitor amplification products, and the lower bands are wild-type DNA PCR products. Each lane represents data from an individual mouse. Sample DNA levels were equalized by using primers for the single-copy mammalian IL-4pr gene. Spirochete DNA was amplified by using primers for ospA . The amount of BC3 competitor spiked into each sample was 0.25 pg.

Techniques Used: Polymerase Chain Reaction, Amplification, Mouse Assay

29) Product Images from "Human heart failure biomarker immunosensor based on excessively tilted fiber gratings"

Article Title: Human heart failure biomarker immunosensor based on excessively tilted fiber gratings

Journal: Biomedical Optics Express

doi: 10.1364/BOE.8.000057

Preparation and purification of NT-proBNP MAbs. (a) Amplification of the NT-proBNP gene by PCR. Lane 1: PCR product from the NT-proBNP gene. (b) Identification of recombinant vector by restriction endonuclease analysis. Lane 1: T3P × 4-pET20b
Figure Legend Snippet: Preparation and purification of NT-proBNP MAbs. (a) Amplification of the NT-proBNP gene by PCR. Lane 1: PCR product from the NT-proBNP gene. (b) Identification of recombinant vector by restriction endonuclease analysis. Lane 1: T3P × 4-pET20b

Techniques Used: Purification, Amplification, Polymerase Chain Reaction, Recombinant, Plasmid Preparation

30) Product Images from "Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients"

Article Title: Impact of Single Nucleotide Polymorphism in IL-4, IL-4R Genes and Systemic Concentration of IL-4 on the Incidence of Glioma in Iraqi Patients

Journal: International Journal of Medical Sciences

doi: 10.7150/ijms.9412

DNA sequencing from portions of the IL-4 gene from the forward strand are shown. The arrow indicates the position of SNP rs2070784 a: represents the genotype CC; b: TC and c: TT.
Figure Legend Snippet: DNA sequencing from portions of the IL-4 gene from the forward strand are shown. The arrow indicates the position of SNP rs2070784 a: represents the genotype CC; b: TC and c: TT.

Techniques Used: DNA Sequencing

Gel electrophoresis for (A) IL-4 PCR product visualized under UV light. M, 1000 bp marker, lane 1-10: from blood of glioma patients, lane 11-14: from blood of control group. The size of product is 280 bp and (B) IL-4R PCR product visualized under UV light. M, 1000 bp marker, lane 1-9: from blood of glioma patients, lane 10-11: from blood of control group. The size of product is 140 bp.
Figure Legend Snippet: Gel electrophoresis for (A) IL-4 PCR product visualized under UV light. M, 1000 bp marker, lane 1-10: from blood of glioma patients, lane 11-14: from blood of control group. The size of product is 280 bp and (B) IL-4R PCR product visualized under UV light. M, 1000 bp marker, lane 1-9: from blood of glioma patients, lane 10-11: from blood of control group. The size of product is 140 bp.

Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker

Mean serum concentration of IL-4 (pg/ml) in glioma patients and control group subjects as described in the materials and methods.
Figure Legend Snippet: Mean serum concentration of IL-4 (pg/ml) in glioma patients and control group subjects as described in the materials and methods.

Techniques Used: Concentration Assay

31) Product Images from "Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic"

Article Title: Diversity of Capsular Polysaccharide Gene Clusters in Kpc-Producing Klebsiella pneumoniae Clinical Isolates of Sequence Type 258 Involved in the Italian Epidemic

Journal: PLoS ONE

doi: 10.1371/journal.pone.0096827

Map showing the distribution of Italian centers from which the 46 KPC-Kp strains of ST258 or ST512 investigated for CPS typing by the modified multiplex PCR were originated, and distribution of the different types of cps gene clusters. Centers were as follows: 01, Milan; 02, Varese; 03, Lecco; 04, Torino; 05, Novara; 06, Genoa; 07, Sanremo; 08, Verona; 09, Bolzano; 10, Modena; 11, Modena; 15, Ancona; 16, Rome; 18, Foggia; 19, Lecce; 20, Naples; 22, Cosenza; 23, Palermo; 24, Catania.
Figure Legend Snippet: Map showing the distribution of Italian centers from which the 46 KPC-Kp strains of ST258 or ST512 investigated for CPS typing by the modified multiplex PCR were originated, and distribution of the different types of cps gene clusters. Centers were as follows: 01, Milan; 02, Varese; 03, Lecco; 04, Torino; 05, Novara; 06, Genoa; 07, Sanremo; 08, Verona; 09, Bolzano; 10, Modena; 11, Modena; 15, Ancona; 16, Rome; 18, Foggia; 19, Lecce; 20, Naples; 22, Cosenza; 23, Palermo; 24, Catania.

Techniques Used: Modification, Multiplex Assay, Polymerase Chain Reaction

32) Product Images from "TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival"

Article Title: TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.449

Xenografts of NB-TIC lines express CD15 and MMP-2 in tumor sections overlapping with or adjacent to TLX. Sections from the xenografts were stained with double immunofluorescence for TLX/CD15 or TLX/MMP-2, and representative images are shown. TLX (red) and CD15/MMP-2 (green). Scale bar represents 50 μ m. Tissue structure is shown by HE staining. Scale bar represents 11 × 200 μ m
Figure Legend Snippet: Xenografts of NB-TIC lines express CD15 and MMP-2 in tumor sections overlapping with or adjacent to TLX. Sections from the xenografts were stained with double immunofluorescence for TLX/CD15 or TLX/MMP-2, and representative images are shown. TLX (red) and CD15/MMP-2 (green). Scale bar represents 50 μ m. Tissue structure is shown by HE staining. Scale bar represents 11 × 200 μ m

Techniques Used: Staining, Immunofluorescence

TLX promotes migration and invasion in IMR-32 cells. ( a ) Invasion and migration assays were performed as described in Materials and Methods using WT IMR-32, shRNA-control (ShCtrl) or Sh2 and Sh3 lines. Values depict the absorbance at 450 nm, representing the invasion/migration index values. ( b ) Graph depicting the increase of secreted MMP-2 levels in the conditioned media of WT, ShCtrl, Sh2 and Sh3 cells measured by ELISA. ( c ) Immunoblot analysis of MMP-2 and MMP-9 from conditioned media of control or shTLX cells. Remaining cells in the plate were lysed and used for GAPDH control. ( d ) Fold change in the MMP-2 and MMP-9 transcript, calculated by normalization against GAPDH in WT or TLX-silenced IMR-32 cells. ( e ) Migration assay in IMR-32 cells as described in ( a ), with the indicated transfections below
Figure Legend Snippet: TLX promotes migration and invasion in IMR-32 cells. ( a ) Invasion and migration assays were performed as described in Materials and Methods using WT IMR-32, shRNA-control (ShCtrl) or Sh2 and Sh3 lines. Values depict the absorbance at 450 nm, representing the invasion/migration index values. ( b ) Graph depicting the increase of secreted MMP-2 levels in the conditioned media of WT, ShCtrl, Sh2 and Sh3 cells measured by ELISA. ( c ) Immunoblot analysis of MMP-2 and MMP-9 from conditioned media of control or shTLX cells. Remaining cells in the plate were lysed and used for GAPDH control. ( d ) Fold change in the MMP-2 and MMP-9 transcript, calculated by normalization against GAPDH in WT or TLX-silenced IMR-32 cells. ( e ) Migration assay in IMR-32 cells as described in ( a ), with the indicated transfections below

Techniques Used: Migration, shRNA, Enzyme-linked Immunosorbent Assay, Transfection

TLX transcriptionally regulates MMP-2 and Oct-4 in hypoxic NB cells. ( a ) Luciferase activity in 293T cells after co-transfection of MMP-2 promoter-luciferase constructs with TLX or control vector. ( b and c ) Top panels depict schematic representation of regions analyzed by ChIP within MMP-2 promoter or Oct-4 promoter ( c ). Occupancy of TLX, Pol-II and H3K9 acetylation across the 1.2 kb upstream regulatory regions of TLX-regulated genes MMP-2 and OCT-4 , and control actin promoter was monitored by ChIP analysis upon normoxia ( b ) or hypoxia ( c ). Chromatin was isolated from normoxia- or hypoxia-treated cells and ChIP analysis was performed as described in Materials and Methods. Amplicon from each immunoprecipitate is represented as the percentage of input. Each error bar indicates standard deviation calculated from triplicates. ( d ) Graph represents the binding of TLX to MMP-2 promoter as a function of absorbance at 450/650 nm. Biotin-labeled consensus oligos were used to capture TLX of nuclear lysate from WT IMR-32. A nonspecific capture oligo served as control, and rabbit IgG were used to exclude nonspecific binding. Mutant oligos (Mut1 or Mut2) were used to confirm the specificity of capture. The values obtained are means of three independent experiments along with S.D. as error bars
Figure Legend Snippet: TLX transcriptionally regulates MMP-2 and Oct-4 in hypoxic NB cells. ( a ) Luciferase activity in 293T cells after co-transfection of MMP-2 promoter-luciferase constructs with TLX or control vector. ( b and c ) Top panels depict schematic representation of regions analyzed by ChIP within MMP-2 promoter or Oct-4 promoter ( c ). Occupancy of TLX, Pol-II and H3K9 acetylation across the 1.2 kb upstream regulatory regions of TLX-regulated genes MMP-2 and OCT-4 , and control actin promoter was monitored by ChIP analysis upon normoxia ( b ) or hypoxia ( c ). Chromatin was isolated from normoxia- or hypoxia-treated cells and ChIP analysis was performed as described in Materials and Methods. Amplicon from each immunoprecipitate is represented as the percentage of input. Each error bar indicates standard deviation calculated from triplicates. ( d ) Graph represents the binding of TLX to MMP-2 promoter as a function of absorbance at 450/650 nm. Biotin-labeled consensus oligos were used to capture TLX of nuclear lysate from WT IMR-32. A nonspecific capture oligo served as control, and rabbit IgG were used to exclude nonspecific binding. Mutant oligos (Mut1 or Mut2) were used to confirm the specificity of capture. The values obtained are means of three independent experiments along with S.D. as error bars

Techniques Used: Luciferase, Activity Assay, Cotransfection, Construct, Plasmid Preparation, Chromatin Immunoprecipitation, Isolation, Amplification, Standard Deviation, Binding Assay, Labeling, Mutagenesis

33) Product Images from "Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells"

Article Title: Comparative analysis of canine monocyte- and bone-marrow-derived dendritic cells

Journal: Veterinary Research

doi: 10.1051/vetres/2010012

Cell morphology of canine MΦ and MoDC. (A, B) MoDC generated from monocytes cultured with human GM-CSF and canine IL-4. (A) Cells with dendritic extensions; (B) cluster of MoDC. (C) MΦ obtained from CD14 + monocytes after 6 days of culture in serum-free medium supplemented with human GM-CSF.
Figure Legend Snippet: Cell morphology of canine MΦ and MoDC. (A, B) MoDC generated from monocytes cultured with human GM-CSF and canine IL-4. (A) Cells with dendritic extensions; (B) cluster of MoDC. (C) MΦ obtained from CD14 + monocytes after 6 days of culture in serum-free medium supplemented with human GM-CSF.

Techniques Used: Generated, Cell Culture

34) Product Images from "Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes"

Article Title: Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

Journal: Eurosurveillance

doi: 10.2807/1560-7917.ES.2018.23.6.17-00672

Multiplex PCR for detection of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 , European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) in the context of animal health and food safety, 2017
Figure Legend Snippet: Multiplex PCR for detection of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 , European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) in the context of animal health and food safety, 2017

Techniques Used: Multiplex Assay, Polymerase Chain Reaction

35) Product Images from "Enhanced Chromatin Accessibility and Recruitment of JUNB Mediate the Sustained IL-4 Expression in NFAT1 Deficient T Helper 2 Cells"

Article Title: Enhanced Chromatin Accessibility and Recruitment of JUNB Mediate the Sustained IL-4 Expression in NFAT1 Deficient T Helper 2 Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022042

Increased DNA demethylation status at the IL-4 promoter of NFAT1 deficient Th2 cells. Th2 cells from WT or NFAT1 KO mice were stimulated with anti-CD3 for 6 h or left without stimulation. DNA methylation state at the IL-4 promoter was analyzed by pyrosequencing ( Fig. S2 ) and ChIP analysis using anti-5mC antibodies specific for methylated DNA at IL-4 promoter (B). FoxP3 and β-actin locus were tested as positive or negative control, respectively for methylated DNA experiment, (C). The relative amount of enriched methylated DNA was assessed by real-time PCR analysis. 10% of the input sample was used for PCR amplification and normalized to the input samples from three independent experiments and * P
Figure Legend Snippet: Increased DNA demethylation status at the IL-4 promoter of NFAT1 deficient Th2 cells. Th2 cells from WT or NFAT1 KO mice were stimulated with anti-CD3 for 6 h or left without stimulation. DNA methylation state at the IL-4 promoter was analyzed by pyrosequencing ( Fig. S2 ) and ChIP analysis using anti-5mC antibodies specific for methylated DNA at IL-4 promoter (B). FoxP3 and β-actin locus were tested as positive or negative control, respectively for methylated DNA experiment, (C). The relative amount of enriched methylated DNA was assessed by real-time PCR analysis. 10% of the input sample was used for PCR amplification and normalized to the input samples from three independent experiments and * P

Techniques Used: Mouse Assay, DNA Methylation Assay, Chromatin Immunoprecipitation, Methylation, Negative Control, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification

Sustained IL-4 expression in NFAT1 deficient Th2 cells. Th2 cells differentiated in vitro from WT or NFAT1 KO mice were stimulated with anti-CD3 (α-CD3) for indicated time periods and IL-4 mRNA levels were measured by quantitative RT-PCR by normalizing with β-actin levels (A). PCR products were visualized on ethidium bromide-stained agarose gels (B). Data shown are the mean ± SEM from four separate experiments and * P
Figure Legend Snippet: Sustained IL-4 expression in NFAT1 deficient Th2 cells. Th2 cells differentiated in vitro from WT or NFAT1 KO mice were stimulated with anti-CD3 (α-CD3) for indicated time periods and IL-4 mRNA levels were measured by quantitative RT-PCR by normalizing with β-actin levels (A). PCR products were visualized on ethidium bromide-stained agarose gels (B). Data shown are the mean ± SEM from four separate experiments and * P

Techniques Used: Expressing, In Vitro, Mouse Assay, Quantitative RT-PCR, Polymerase Chain Reaction, Staining

SATB1 and JUNB synergistically transactivate IL-4 promoter with other cofactors. (A) IL-4 reporter construct was transfected into HEK cells in the presence of different amount (0, 0.2 µg (+) or 0.4 (++) µg) of SATB1 or JunB expression plasmid alone or both of them, and then luciferase activity was measured. In each transfection, 0.1μg of TK-luciferase plasmid was added as an internal control for normalization of transfection efficiency. Transfected cells were harvested after 24 h in reporter lysis buffer, and analyzed for luciferase activity. (B) IL-4 reporter construct was transfected into HEK cells, in duplicate, with 0.2 µg of STATB1/JunB expression plasmids, in the absence or presence of 0.2 µg of p300 or/and PCAF expression plasmids. In each transfection, 0.1 µg of TK-luciferase plasmid was added and total DNA was maintained at 0.8 µg by addition of the appropriate amounts of pcDNA3 control plasmid. After 24 h, cells were harvested and analyzed for firefly and Renilla luciferase activities. Values were normalized to Renilla activities. The graphs in A-B represents mean ± SEM, n = 3 and * P
Figure Legend Snippet: SATB1 and JUNB synergistically transactivate IL-4 promoter with other cofactors. (A) IL-4 reporter construct was transfected into HEK cells in the presence of different amount (0, 0.2 µg (+) or 0.4 (++) µg) of SATB1 or JunB expression plasmid alone or both of them, and then luciferase activity was measured. In each transfection, 0.1μg of TK-luciferase plasmid was added as an internal control for normalization of transfection efficiency. Transfected cells were harvested after 24 h in reporter lysis buffer, and analyzed for luciferase activity. (B) IL-4 reporter construct was transfected into HEK cells, in duplicate, with 0.2 µg of STATB1/JunB expression plasmids, in the absence or presence of 0.2 µg of p300 or/and PCAF expression plasmids. In each transfection, 0.1 µg of TK-luciferase plasmid was added and total DNA was maintained at 0.8 µg by addition of the appropriate amounts of pcDNA3 control plasmid. After 24 h, cells were harvested and analyzed for firefly and Renilla luciferase activities. Values were normalized to Renilla activities. The graphs in A-B represents mean ± SEM, n = 3 and * P

Techniques Used: Construct, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Lysis

Identification and analysis of IL-4 promoter-binding transcription factors. EMSA assay was carried out with nuclear extracts prepared from Th2 cells of WT and NFAT1 KO or from Jurkat cells that were stimulated with anti-CD3 for 6 h or PMA/I for 2 h, respectively (A). 32 P-labelled P2 probes were incubated with indicated nuclear extracts. IκB probe containing κB element was used as a positive control and 100 pmol unlabeled iκB probe was used as a competitor to interfere the complex formation. (−) or (+) indicates in the absence or presence competitor, respectively. The DNA binding complexes that appeared or were enhanced after stimulation are indicated. Arrows indicate the identified proteins that bound to the corresponding gel band. Equal loading of nuclear extracts was controlled by using the binding site for the constitutive factor EF-1 as a probe. Identification of DNA-binding transcription factors was carried out by micro-LC/LC-MS/MS analysis as described in Material and Methods. (B) Predicted DNA binding elements for the P2 region of IL-4 promoter.
Figure Legend Snippet: Identification and analysis of IL-4 promoter-binding transcription factors. EMSA assay was carried out with nuclear extracts prepared from Th2 cells of WT and NFAT1 KO or from Jurkat cells that were stimulated with anti-CD3 for 6 h or PMA/I for 2 h, respectively (A). 32 P-labelled P2 probes were incubated with indicated nuclear extracts. IκB probe containing κB element was used as a positive control and 100 pmol unlabeled iκB probe was used as a competitor to interfere the complex formation. (−) or (+) indicates in the absence or presence competitor, respectively. The DNA binding complexes that appeared or were enhanced after stimulation are indicated. Arrows indicate the identified proteins that bound to the corresponding gel band. Equal loading of nuclear extracts was controlled by using the binding site for the constitutive factor EF-1 as a probe. Identification of DNA-binding transcription factors was carried out by micro-LC/LC-MS/MS analysis as described in Material and Methods. (B) Predicted DNA binding elements for the P2 region of IL-4 promoter.

Techniques Used: Binding Assay, Incubation, Positive Control, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

In vivo binding of JUNB, SATB1 and cofactors to the IL-4 promoter. Th2 cells from WT or NFAT1 KO mice were stimulated with anti-CD3 for 6 h or left without stimulation. The relative amount of DNA-protein complex enriched at the P2 locus of IL-4 promoter was analyzed by ChIP using control normal IgG (F) or specific antibodies for JUNB (A), SATB1 (B), and other cofactors such as P300 (C), PCAF (D) and HDAC1 (E). Relative enrichment at the P2 locus of IL-4 promoter in the precipitated samples compared to total chromatin (input) is shown. Data are representative of three independent experiments and * P
Figure Legend Snippet: In vivo binding of JUNB, SATB1 and cofactors to the IL-4 promoter. Th2 cells from WT or NFAT1 KO mice were stimulated with anti-CD3 for 6 h or left without stimulation. The relative amount of DNA-protein complex enriched at the P2 locus of IL-4 promoter was analyzed by ChIP using control normal IgG (F) or specific antibodies for JUNB (A), SATB1 (B), and other cofactors such as P300 (C), PCAF (D) and HDAC1 (E). Relative enrichment at the P2 locus of IL-4 promoter in the precipitated samples compared to total chromatin (input) is shown. Data are representative of three independent experiments and * P

Techniques Used: In Vivo, Binding Assay, Mouse Assay, Chromatin Immunoprecipitation

NFAT1 deficient Th2 cells have more permissive chromatin structure at the IL-4 promoter. (A) For chromatin accessibility analysis, Th2 cells differentiated in vitro from WT or NFAT1 KO mice were stimulated with anti-CD3 for 6 h. Nuclei isolated from each group and were incubated with or without 50 U Mnase. Fifty nanograms of genomic DNA from each treatment were subjected to real-time PCR analysis with indicated primer sets covering IL-4 promoter. The Ct values generated were converted to DNA concentrations by using the standard curve. MNase accessibility was expressed as a relative value of undigested genomic DNA and plotted for each primer set. Graphs depict the PCR products from digested samples normalized to the PCR products from undigested samples and show mean ± SEM, n = 3 and ** P
Figure Legend Snippet: NFAT1 deficient Th2 cells have more permissive chromatin structure at the IL-4 promoter. (A) For chromatin accessibility analysis, Th2 cells differentiated in vitro from WT or NFAT1 KO mice were stimulated with anti-CD3 for 6 h. Nuclei isolated from each group and were incubated with or without 50 U Mnase. Fifty nanograms of genomic DNA from each treatment were subjected to real-time PCR analysis with indicated primer sets covering IL-4 promoter. The Ct values generated were converted to DNA concentrations by using the standard curve. MNase accessibility was expressed as a relative value of undigested genomic DNA and plotted for each primer set. Graphs depict the PCR products from digested samples normalized to the PCR products from undigested samples and show mean ± SEM, n = 3 and ** P

Techniques Used: In Vitro, Mouse Assay, Isolation, Incubation, Real-time Polymerase Chain Reaction, Generated, Polymerase Chain Reaction

36) Product Images from "Splice variants and seasonal expression of buffalo HSF genes"

Article Title: Splice variants and seasonal expression of buffalo HSF genes

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-014-0563-y

Buffalo HSF-1, 2, 4, and 5 gene sequence
Figure Legend Snippet: Buffalo HSF-1, 2, 4, and 5 gene sequence

Techniques Used: Sequencing

Detection of the buffalo HSF gene splice variant in tissues. a RT-PCR was performed for HSF-1 gene between exon 9 and 13, resulting in the amplification of a 273 bp fragment, corresponding to the original cloned product called “HSF-1Tv1”
Figure Legend Snippet: Detection of the buffalo HSF gene splice variant in tissues. a RT-PCR was performed for HSF-1 gene between exon 9 and 13, resulting in the amplification of a 273 bp fragment, corresponding to the original cloned product called “HSF-1Tv1”

Techniques Used: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Clone Assay

Multiple sequence alignment of the identified heat shock factor sequences. Buffalo HSF-1, HSF-2, HSF-4 and HSF-5 protein sequences were aligned by using cluatalW2 program in BioEdit software. The secondary structure of HSF protein sequence is shown above
Figure Legend Snippet: Multiple sequence alignment of the identified heat shock factor sequences. Buffalo HSF-1, HSF-2, HSF-4 and HSF-5 protein sequences were aligned by using cluatalW2 program in BioEdit software. The secondary structure of HSF protein sequence is shown above

Techniques Used: Sequencing, Software

Seasonal differences in expression of buffalo HSF-1, 4, and 5 genes. ( a – d ) RT-qPCR analysis of HSF-1, 4, and 5 transcripts was performed in three different seasons as indicated. The level of transcripts from the gene encoding ribosomal protein
Figure Legend Snippet: Seasonal differences in expression of buffalo HSF-1, 4, and 5 genes. ( a – d ) RT-qPCR analysis of HSF-1, 4, and 5 transcripts was performed in three different seasons as indicated. The level of transcripts from the gene encoding ribosomal protein

Techniques Used: Expressing, Quantitative RT-PCR

Distinct tissue specific expression of buffalo HSF-1, 2, 4, and 5 genes ( a – d ). RT-qPCR analysis of HSF-1, 2, 4, and 5 transcripts in the indicated tissues was performed. The level of transcripts from the gene encoding ribosomal protein RPS 18
Figure Legend Snippet: Distinct tissue specific expression of buffalo HSF-1, 2, 4, and 5 genes ( a – d ). RT-qPCR analysis of HSF-1, 2, 4, and 5 transcripts in the indicated tissues was performed. The level of transcripts from the gene encoding ribosomal protein RPS 18

Techniques Used: Expressing, Quantitative RT-PCR

37) Product Images from "Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans"

Article Title: Bidirectional regulation of thermotaxis by glutamate transmissions in Caenorhabditis elegans

Journal: The EMBO Journal

doi: 10.1038/emboj.2011.13

Expression pattern of EAT-4. ( A ) The full-length eat-4∷gfp fusion gene with 5.5 kb of promoter region. ( B , C ) The expression of full-length eat-4∷gfp in the head of wild-type animals ( B ) and of unc-104(e1265) mutants ( C ). Anterior is to the left and dorsal is on the top. ( B ) Fluorescence was observed in nerve ring and cell body of many neurons including AFD, AWC (left panel), RIA (top-right panel), and AIZ neurons (bottom-right panel) in wild type. ( C ) Fluorescence was not observed in the nerve ring but in the cell body of many neurons, including AFD and AWC in unc-104(e1265) mutants. ( D ) The proposed thermotaxis neural circuit ( Mori and Ohshima, 1995 ; Kuhara et al, 2008 ). Temperature is sensed by AFD and AWC sensory neurons, thermal information from AFD and AWC is conveyed to AIY interneuron, and the subsequent neural information from AIY is further conveyed to AIZ and RIA interneuron. Neurons expressing the full-length eat-4∷gfp are coloured green.
Figure Legend Snippet: Expression pattern of EAT-4. ( A ) The full-length eat-4∷gfp fusion gene with 5.5 kb of promoter region. ( B , C ) The expression of full-length eat-4∷gfp in the head of wild-type animals ( B ) and of unc-104(e1265) mutants ( C ). Anterior is to the left and dorsal is on the top. ( B ) Fluorescence was observed in nerve ring and cell body of many neurons including AFD, AWC (left panel), RIA (top-right panel), and AIZ neurons (bottom-right panel) in wild type. ( C ) Fluorescence was not observed in the nerve ring but in the cell body of many neurons, including AFD and AWC in unc-104(e1265) mutants. ( D ) The proposed thermotaxis neural circuit ( Mori and Ohshima, 1995 ; Kuhara et al, 2008 ). Temperature is sensed by AFD and AWC sensory neurons, thermal information from AFD and AWC is conveyed to AIY interneuron, and the subsequent neural information from AIY is further conveyed to AIZ and RIA interneuron. Neurons expressing the full-length eat-4∷gfp are coloured green.

Techniques Used: Expressing, Fluorescence

38) Product Images from "The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription"

Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

Journal: Virology Journal

doi: 10.1186/s12985-016-0600-9

The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P
Figure Legend Snippet: The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Injection

UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P
Figure Legend Snippet: UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P

Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase

Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box
Figure Legend Snippet: Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box

Techniques Used: Mutagenesis, Labeling, Amplification, Expressing, Infection, Polymerase Chain Reaction

The mutated UL7 gene modulates viral proliferation. a Dynamic profile of the UL7-MU viral strain at 37 °C. Vero cells were infected with UL7-MU (filled circles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells. b Morphological features of UL7-MU mutant plaques at 37 °C. Vero cells were infected with UL7-MU or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profile of the UL7-MU viral strain at 34 °C. Vero cells were infected with UL7-MU (filled triangles) or HSV-1 WT (open triangles) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells
Figure Legend Snippet: The mutated UL7 gene modulates viral proliferation. a Dynamic profile of the UL7-MU viral strain at 37 °C. Vero cells were infected with UL7-MU (filled circles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells. b Morphological features of UL7-MU mutant plaques at 37 °C. Vero cells were infected with UL7-MU or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profile of the UL7-MU viral strain at 34 °C. Vero cells were infected with UL7-MU (filled triangles) or HSV-1 WT (open triangles) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells

Techniques Used: Infection, Mutagenesis, Staining

The UL7-MU viral strain exhibits lower virulence in infected mice compared with the WT strain. a BALB/c mice were infected via intranasal instillation of 2.3 × 10 5 PFU of WT HSV-1 (open boxes), UL7-MU (filled circles), MU-complemented P1 (filled triangles) or PBS (open rhombuses) as a control. The weights of the mice were measured every 2 days. Data are shown as means ± SD. ∗ P
Figure Legend Snippet: The UL7-MU viral strain exhibits lower virulence in infected mice compared with the WT strain. a BALB/c mice were infected via intranasal instillation of 2.3 × 10 5 PFU of WT HSV-1 (open boxes), UL7-MU (filled circles), MU-complemented P1 (filled triangles) or PBS (open rhombuses) as a control. The weights of the mice were measured every 2 days. Data are shown as means ± SD. ∗ P

Techniques Used: Infection, Mouse Assay

The mutated phenotype of UL7-MU is complemented by expression of the UL7 gene in cells. a The lysates of Vero cells transfected with the pcDNA3-UL7 plasmid were separated on a 12 % polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with UL7 antibody. The position of the UL7 protein is indicated. b Morphological features of plaques formed by the complemented UL7-MU and WT viral strains. Cells were infected at 24 h post-transfection with UL7-MU or WT virus at an MOI of 1. The supernatants and cells of the complemented virus were harvested after CPE was observed, followed by plaque assays. Vero cells were infected with MU-complemented P1, WT-control P1, MU-complemented P2, WT-control P2 or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profiles of the MU-complemented P1 and MU-complemented P2 viral strains at 37 °C. Vero cells were infected with the MU-complemented P1 (filled circles), MU-complemented P2 (filled triangles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells
Figure Legend Snippet: The mutated phenotype of UL7-MU is complemented by expression of the UL7 gene in cells. a The lysates of Vero cells transfected with the pcDNA3-UL7 plasmid were separated on a 12 % polyacrylamide gel, transferred to a PVDF membrane, and immunoblotted with UL7 antibody. The position of the UL7 protein is indicated. b Morphological features of plaques formed by the complemented UL7-MU and WT viral strains. Cells were infected at 24 h post-transfection with UL7-MU or WT virus at an MOI of 1. The supernatants and cells of the complemented virus were harvested after CPE was observed, followed by plaque assays. Vero cells were infected with MU-complemented P1, WT-control P1, MU-complemented P2, WT-control P2 or HSV-1 WT at an MOI of 0.00001. The cells were fixed and stained 3 d p.i. c Dynamic profiles of the MU-complemented P1 and MU-complemented P2 viral strains at 37 °C. Vero cells were infected with the MU-complemented P1 (filled circles), MU-complemented P2 (filled triangles) or HSV-1 WT (open boxes) at an MOI of 1. The supernatants and cells were harvested at the indicated time points, and the lysates were titered on Vero cells

Techniques Used: Expressing, Transfection, Plasmid Preparation, Infection, Staining

39) Product Images from "Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells"

Article Title: Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0084340

Comparison of Mcp-2 and Mcp-4 protein levels in mast cells from B6, B6-cma, and DBA/2 mice. Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. A. Cell extracts were resolved on 12.5% SDS gels followed by Coomassie blue staining (top panel) or western blotting with indicated antibodies. B. Cells were subjected to Wright-Giemsa staining (top panel) or immunofluorescent staining with indicated antibodies.
Figure Legend Snippet: Comparison of Mcp-2 and Mcp-4 protein levels in mast cells from B6, B6-cma, and DBA/2 mice. Mast cells were derived from bone marrow (BM) and peritoneal cavity (PC) of B6, B6-cma, and DBA/2 mice. A. Cell extracts were resolved on 12.5% SDS gels followed by Coomassie blue staining (top panel) or western blotting with indicated antibodies. B. Cells were subjected to Wright-Giemsa staining (top panel) or immunofluorescent staining with indicated antibodies.

Techniques Used: Mouse Assay, Derivative Assay, Staining, Western Blot

Identification of markedly increased expressions of Mcp-2 and Mcp-4 in BMMCs from a subpopulation of JAK2V617F transgenic mice. A. Detection of a predominant protein band in cell extracts of BMMCs from a variant line of mice. BMMCs from two JAK2V617F transgenic mice were extracted in a buffer containing 1% Triton X-100 or 1X SDS gel sample buffer were resolved on 10% SDS gel, and proteins were visualized by Coomassie blue staining. The arrow points to a predominant band. B. Verification of Mcp-2 and Mcp-4 over-expressions by Western blotting with specific antibodies. Extracts of BMMCs were separated on 12.5% SDS gel and subjected to Western blotting analyses with anti-Mcp-2 and Mcp-4. C. Verification of Mcp-2 and Mcp-4 over-expression by immunofluorescent cell staining. Mcp-2 and Mcp-4 were probed with specific antibodies followed by Cy-3-conjugated secondary antibodies (red). The nuclei (blue) were revealed by staining with Hoechst 33258.
Figure Legend Snippet: Identification of markedly increased expressions of Mcp-2 and Mcp-4 in BMMCs from a subpopulation of JAK2V617F transgenic mice. A. Detection of a predominant protein band in cell extracts of BMMCs from a variant line of mice. BMMCs from two JAK2V617F transgenic mice were extracted in a buffer containing 1% Triton X-100 or 1X SDS gel sample buffer were resolved on 10% SDS gel, and proteins were visualized by Coomassie blue staining. The arrow points to a predominant band. B. Verification of Mcp-2 and Mcp-4 over-expressions by Western blotting with specific antibodies. Extracts of BMMCs were separated on 12.5% SDS gel and subjected to Western blotting analyses with anti-Mcp-2 and Mcp-4. C. Verification of Mcp-2 and Mcp-4 over-expression by immunofluorescent cell staining. Mcp-2 and Mcp-4 were probed with specific antibodies followed by Cy-3-conjugated secondary antibodies (red). The nuclei (blue) were revealed by staining with Hoechst 33258.

Techniques Used: Transgenic Assay, Mouse Assay, Variant Assay, SDS-Gel, Staining, Western Blot, Over Expression

Identification of Mcp-2 and Mcp-4 gene variants. Schematic alignment of amino acid sequences (A.) and promoter region DNA sequences (B.) of Mcp-2P and Mcp-4P from control B6 and variant B6-cma mice. Variant amino acid residues and nucleotide bases are highlighted in bold. A vertical line “|” denotes identical amino acids or bases, and a dash “–” stands for deletions. Putative Mitf binding consensus motifs (CANNTG E-boxes) and an NdeI restriction cleavage site (CATATG) in the variant form of Mcp-2 or Mcp-4 are underlined. C. Detection of Mcp-2 and Mcp-4 gene variations in the promoter regions by allele-specific PCR and NdeI restriction fragment length polymorphism (RFLP), respectively. Normal form of Mcp-2P was detected by PCR with primers 5′-ctcacactggtcaacacaaacatta and 5′-tctgctgttaaacacaaacacagtct, while the Mcp-2P variant was amplified by using primers 5′-ctcacactggtcaacacaaacattg and 5′-tctgctgttaaacacaaacacagtca. The expected product size for both is 131 bp. The variant form of Mcp-4P was revealed by NdeI digestion which gave rise to two fragments while the normal form of Mcp-4P was not cleaved. Data show results for both homozygous and heterozygous mice.
Figure Legend Snippet: Identification of Mcp-2 and Mcp-4 gene variants. Schematic alignment of amino acid sequences (A.) and promoter region DNA sequences (B.) of Mcp-2P and Mcp-4P from control B6 and variant B6-cma mice. Variant amino acid residues and nucleotide bases are highlighted in bold. A vertical line “|” denotes identical amino acids or bases, and a dash “–” stands for deletions. Putative Mitf binding consensus motifs (CANNTG E-boxes) and an NdeI restriction cleavage site (CATATG) in the variant form of Mcp-2 or Mcp-4 are underlined. C. Detection of Mcp-2 and Mcp-4 gene variations in the promoter regions by allele-specific PCR and NdeI restriction fragment length polymorphism (RFLP), respectively. Normal form of Mcp-2P was detected by PCR with primers 5′-ctcacactggtcaacacaaacatta and 5′-tctgctgttaaacacaaacacagtct, while the Mcp-2P variant was amplified by using primers 5′-ctcacactggtcaacacaaacattg and 5′-tctgctgttaaacacaaacacagtca. The expected product size for both is 131 bp. The variant form of Mcp-4P was revealed by NdeI digestion which gave rise to two fragments while the normal form of Mcp-4P was not cleaved. Data show results for both homozygous and heterozygous mice.

Techniques Used: Variant Assay, Mouse Assay, Binding Assay, Polymerase Chain Reaction, Amplification

Association of Mcp-2 and Mcp-4 overexpressions in BMMCs with gene variants. A. Mice were genotyped for the Mcp-2 gene variant by allele-specific PCR and the Mcp-4 gene variant by restriction fragment length polymorphism (RFLP) with NdeI. BMMCs derived from these mice were analyzed for Mcp-2 and Mcp-4 protein expressions by Commassie blue staining and western blotting. B. BMMCs from B6 and B6-cma mice were analyzed for chymase activity. In at least 45 mice analyzed, there is a perfect correlation of Mcp-2 and Mcp-4 gene variants with overexpression of Mcp-2 and Mcp-4 and increased chymase activity.
Figure Legend Snippet: Association of Mcp-2 and Mcp-4 overexpressions in BMMCs with gene variants. A. Mice were genotyped for the Mcp-2 gene variant by allele-specific PCR and the Mcp-4 gene variant by restriction fragment length polymorphism (RFLP) with NdeI. BMMCs derived from these mice were analyzed for Mcp-2 and Mcp-4 protein expressions by Commassie blue staining and western blotting. B. BMMCs from B6 and B6-cma mice were analyzed for chymase activity. In at least 45 mice analyzed, there is a perfect correlation of Mcp-2 and Mcp-4 gene variants with overexpression of Mcp-2 and Mcp-4 and increased chymase activity.

Techniques Used: Mouse Assay, Variant Assay, Polymerase Chain Reaction, Derivative Assay, Staining, Western Blot, Activity Assay, Over Expression

40) Product Images from "Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum"

Article Title: Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

Journal: BMC Genomics

doi: 10.1186/1471-2164-13-678

The 3 ′ end of A. phagocytophilum virB6 - 4 genes is composed of an unusually large tandem repeat region, which exhibits dramatic variability among strains. A . Map of the human HZ strain virB6 - 3 and virB6 - 4 genes, highlighting the location and structure of several repeat regions (R1-R4). The most variability occurred in R4; this region is 5.88 kb larger than previously reported for the Ap HZ genome (CP000235). The original sequence is diagrammed above the map, with the dashed line representing the segment missing in CP000235. Larger repeated R4 segments of 2.7 kb and 1.15 kb are indicated above. Vertical black bars within each gene designate segments encoding predicted transmembrane domains. BamHI sites, of which there is one in all R4 type 2 repeats (see Figure S2B), are indicated. Also shown are the positions of PCR primers used in C. B . BamHI genomic maps depicting the virB6 - 4 locus (black arrows).The segment encompassing R4 is highlighted below each respective map. In the regions outside the virB6 - 4 locus, corresponding BamHI fragments are shown in the same color. Overall, the optical map sizes were in good agreement with the actual sizes, except within R4. This is attributed to the limitation of optical mapping in resolving fragments
Figure Legend Snippet: The 3 ′ end of A. phagocytophilum virB6 - 4 genes is composed of an unusually large tandem repeat region, which exhibits dramatic variability among strains. A . Map of the human HZ strain virB6 - 3 and virB6 - 4 genes, highlighting the location and structure of several repeat regions (R1-R4). The most variability occurred in R4; this region is 5.88 kb larger than previously reported for the Ap HZ genome (CP000235). The original sequence is diagrammed above the map, with the dashed line representing the segment missing in CP000235. Larger repeated R4 segments of 2.7 kb and 1.15 kb are indicated above. Vertical black bars within each gene designate segments encoding predicted transmembrane domains. BamHI sites, of which there is one in all R4 type 2 repeats (see Figure S2B), are indicated. Also shown are the positions of PCR primers used in C. B . BamHI genomic maps depicting the virB6 - 4 locus (black arrows).The segment encompassing R4 is highlighted below each respective map. In the regions outside the virB6 - 4 locus, corresponding BamHI fragments are shown in the same color. Overall, the optical map sizes were in good agreement with the actual sizes, except within R4. This is attributed to the limitation of optical mapping in resolving fragments

Techniques Used: Sequencing, Polymerase Chain Reaction

41) Product Images from "Claudin-4 is required for vasculogenic mimicry formation in human breast cancer cells"

Article Title: Claudin-4 is required for vasculogenic mimicry formation in human breast cancer cells

Journal: Oncotarget

doi:

Expression of claudin-2, -3, -4, -6, -7, and -17 proteins in HUVEC, MDA-MB-231, and MCF-7 cells HUVEC, MDA-MB-231, and MCF-7 cells were plated on matrigel for 72 h. Western blot analysis of claudin proteins was performed using whole cell protein lysate. (a) Representative blots of claudin-2, -3, -4, -6, -7, and -17 (b) The corresponding expression levels are shown as bar graphs. Claudin protein levels in HUVEC cells were defined as 1. Data represent the mean + SD (n=3), *: p
Figure Legend Snippet: Expression of claudin-2, -3, -4, -6, -7, and -17 proteins in HUVEC, MDA-MB-231, and MCF-7 cells HUVEC, MDA-MB-231, and MCF-7 cells were plated on matrigel for 72 h. Western blot analysis of claudin proteins was performed using whole cell protein lysate. (a) Representative blots of claudin-2, -3, -4, -6, -7, and -17 (b) The corresponding expression levels are shown as bar graphs. Claudin protein levels in HUVEC cells were defined as 1. Data represent the mean + SD (n=3), *: p

Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot

Kinetics of vascular channel formation in HUVEC, MDA-MB-231, and MCF-7 cells (a) HUVEC, MDA-MB-231, and MCF-7 cells were plated on matrigel, and images were acquired using phase-contrast microscopy at the times indicated. HUVEC cells started to form vascular channels 6 h after plating whereas MDA-MB-231 cells formed vascular channels at 24 h. Well-defined patterned networks were observed by 72 h. MCF-7 cells failed to form patterned networks. (b) Periodic Acid-Schiff (PAS) staining of HUVEC and MDA-MB-231 cells plated on matrigel for 72 h to identify secreted extracellular matrix. Pink staining identifies glycogen and related mucopolysaccharides secreted by cells to form the extracellular matrix-rich vascular channels.
Figure Legend Snippet: Kinetics of vascular channel formation in HUVEC, MDA-MB-231, and MCF-7 cells (a) HUVEC, MDA-MB-231, and MCF-7 cells were plated on matrigel, and images were acquired using phase-contrast microscopy at the times indicated. HUVEC cells started to form vascular channels 6 h after plating whereas MDA-MB-231 cells formed vascular channels at 24 h. Well-defined patterned networks were observed by 72 h. MCF-7 cells failed to form patterned networks. (b) Periodic Acid-Schiff (PAS) staining of HUVEC and MDA-MB-231 cells plated on matrigel for 72 h to identify secreted extracellular matrix. Pink staining identifies glycogen and related mucopolysaccharides secreted by cells to form the extracellular matrix-rich vascular channels.

Techniques Used: Multiple Displacement Amplification, Microscopy, Staining

Effects of claudin blocking antibodies on cell proliferation, morphology, and tubule formation (a) MTT assay was used to assess the effects of different blocking antibodies on the proliferation of HUVEC and MDA-MB-231 cells. HUVEC and MDA-MB-231 cells were plated in 96-well plates. Medium containing claudin-2, -3, -4, -6, -7, or -17 blocking antibodies (1 μg/mL) were added to the 96-well plates. An equivalent volume of mouse IgG1 control antibody was used as a control. Data represent the mean + SD (n=3). *: p
Figure Legend Snippet: Effects of claudin blocking antibodies on cell proliferation, morphology, and tubule formation (a) MTT assay was used to assess the effects of different blocking antibodies on the proliferation of HUVEC and MDA-MB-231 cells. HUVEC and MDA-MB-231 cells were plated in 96-well plates. Medium containing claudin-2, -3, -4, -6, -7, or -17 blocking antibodies (1 μg/mL) were added to the 96-well plates. An equivalent volume of mouse IgG1 control antibody was used as a control. Data represent the mean + SD (n=3). *: p

Techniques Used: Blocking Assay, MTT Assay, Multiple Displacement Amplification

Analysis of vascular channel formation following stable transfection of MDA-MB-231 cells with claudin-4-specific shRNA plasmids or lentiviral particles MDA-MB-231 cells grown to 60% confluence were transfected with claudin-4 specific plasmids, lentiviral particles, or control shRNAs. Transduced cells were selected with puromycin (2 μg/ml) after 24 h. (a) Levels of claudin-4 mRNA were examined by nested reverse-transcription-polymerase chain reaction (RT-PCR). Claudin-4 mRNA levels were determined by densitometry and normalized to GAPDH . Changes are expressed as a percentage of control. #: p
Figure Legend Snippet: Analysis of vascular channel formation following stable transfection of MDA-MB-231 cells with claudin-4-specific shRNA plasmids or lentiviral particles MDA-MB-231 cells grown to 60% confluence were transfected with claudin-4 specific plasmids, lentiviral particles, or control shRNAs. Transduced cells were selected with puromycin (2 μg/ml) after 24 h. (a) Levels of claudin-4 mRNA were examined by nested reverse-transcription-polymerase chain reaction (RT-PCR). Claudin-4 mRNA levels were determined by densitometry and normalized to GAPDH . Changes are expressed as a percentage of control. #: p

Techniques Used: Stable Transfection, Multiple Displacement Amplification, shRNA, Transfection, Reverse Transcription Polymerase Chain Reaction

42) Product Images from "Construction of CTLA-4-Ig Fusion Gene in pBudCE4.1 Expression Vector"

Article Title: Construction of CTLA-4-Ig Fusion Gene in pBudCE4.1 Expression Vector

Journal: Avicenna Journal of Medical Biotechnology

doi:

ctla-4 gene PCR product with Pfu polymerase enzyme M: 1 kb ladder 1: PCR product using specific primers (CTLA4- FOR / CTLA4-fuse).
Figure Legend Snippet: ctla-4 gene PCR product with Pfu polymerase enzyme M: 1 kb ladder 1: PCR product using specific primers (CTLA4- FOR / CTLA4-fuse).

Techniques Used: Polymerase Chain Reaction

43) Product Images from "The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription"

Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

Journal: Virology Journal

doi: 10.1186/s12985-016-0600-9

UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P
Figure Legend Snippet: UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P

Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase

44) Product Images from "Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region"

Article Title: Down-regulation of interferon regulatory factor 4 gene expression in leukemic cells due to hypermethylation of CpG motifs in the promoter region

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki1001

Methylation pattern of CpG sites in the IRF-4 promoter region. Methylation is detected via sequencing of bisulfite-treated DNA from hematopoietic cells. ( A ) Schematic figure of the IRF-4 promoter region (see Figure 3A ) for each cell line. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream), white circles mean no methylated clone (from eight), gray circles mean one to four methylated clones and black circles mean five to eight methylated clones. IRF-4 expression is denoted on the right. Below, CpG sites maybe responsible for methylation-dependent IRF-4 silencing (arrows); R1, region with generally high methylation; R2, region with correlation between methylation and IRF-4 expression; R3, region with generally low methylation. Putative binding sites for transcription factors are shown above (5× AP1, 3× Sp1, 2× Ets-1, 2× κB, 1× NF-3, 1× CD28RE, 1× PU.1). ( B ) Schematic figure of specific region 2 (R2): CpG sites from #10 to #22 are shown above. Number of methylated clones (from eight) is shown for each cell line.
Figure Legend Snippet: Methylation pattern of CpG sites in the IRF-4 promoter region. Methylation is detected via sequencing of bisulfite-treated DNA from hematopoietic cells. ( A ) Schematic figure of the IRF-4 promoter region (see Figure 3A ) for each cell line. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream), white circles mean no methylated clone (from eight), gray circles mean one to four methylated clones and black circles mean five to eight methylated clones. IRF-4 expression is denoted on the right. Below, CpG sites maybe responsible for methylation-dependent IRF-4 silencing (arrows); R1, region with generally high methylation; R2, region with correlation between methylation and IRF-4 expression; R3, region with generally low methylation. Putative binding sites for transcription factors are shown above (5× AP1, 3× Sp1, 2× Ets-1, 2× κB, 1× NF-3, 1× CD28RE, 1× PU.1). ( B ) Schematic figure of specific region 2 (R2): CpG sites from #10 to #22 are shown above. Number of methylated clones (from eight) is shown for each cell line.

Techniques Used: Methylation, Sequencing, Labeling, Clone Assay, Expressing, Binding Assay

Investigation of putative mechanisms for IRF-4 deregulation. ( A–C ) Influence of in vitro methylation on the activity of an IRF-4 promoter-reporter gene construct. (A) Control of complete methylation via restriction. Digestion of the construct before (lanes 2, 4 and 6) and after methylation (lanes 1, 3 and 5) with respective methylation-sensitive (HpaII, lanes 1 and 2) and -resistant endonucleases (MspI, lanes 3 and 4). (B and C) Reporter gene assays with non-methylated control (co) and in vitro methylated (met) IRF-4 promoter constructs and SD-1 (B) or Jurkat cells (C). The promoter activity is displayed as fold increase to non-methylated construct (via ratio of firefly to renilla luciferase). ( D ) Expression of DNMT and MBP mRNA in various hematopoietic cells. RT–PCR of various hematopoietic cells: SD-1 (lane 1), RPMI-8226 (lane 2), BV-173 (lane 3), U-937 (lane 4), CML-T1 (lane 5), LAMA-84 (lane 6), Jurkat (lane 7), EM-2 (lane 8) and K-562 (lane 9). β-Actin is used as reference.
Figure Legend Snippet: Investigation of putative mechanisms for IRF-4 deregulation. ( A–C ) Influence of in vitro methylation on the activity of an IRF-4 promoter-reporter gene construct. (A) Control of complete methylation via restriction. Digestion of the construct before (lanes 2, 4 and 6) and after methylation (lanes 1, 3 and 5) with respective methylation-sensitive (HpaII, lanes 1 and 2) and -resistant endonucleases (MspI, lanes 3 and 4). (B and C) Reporter gene assays with non-methylated control (co) and in vitro methylated (met) IRF-4 promoter constructs and SD-1 (B) or Jurkat cells (C). The promoter activity is displayed as fold increase to non-methylated construct (via ratio of firefly to renilla luciferase). ( D ) Expression of DNMT and MBP mRNA in various hematopoietic cells. RT–PCR of various hematopoietic cells: SD-1 (lane 1), RPMI-8226 (lane 2), BV-173 (lane 3), U-937 (lane 4), CML-T1 (lane 5), LAMA-84 (lane 6), Jurkat (lane 7), EM-2 (lane 8) and K-562 (lane 9). β-Actin is used as reference.

Techniques Used: In Vitro, Methylation, Activity Assay, Construct, Luciferase, Expressing, Reverse Transcription Polymerase Chain Reaction

Expression of IRF-4 in hematopoietic cells after treatment with AzadC. Representative cells and experiments are shown. ( A ) RT–PCR after incubation of CML-T1 and EM-2 with 0.5, 1 and 3 µM AzadC for 72 h; ( B ) RT–PCR after treatment of CML-T1 and EM-2 with 3 µM AzadC for 24, 48 and 72 h; ( C ) immunoblotting after treatment of CML-T1 and LAMA-84 with 1 and 3 µM AzadC for 72 h; ( D ) RT–PCR after treatment of BV-173 for 24, 48 and 72 h and RPMI-8226 for 72 h with 3 µM AzadC.
Figure Legend Snippet: Expression of IRF-4 in hematopoietic cells after treatment with AzadC. Representative cells and experiments are shown. ( A ) RT–PCR after incubation of CML-T1 and EM-2 with 0.5, 1 and 3 µM AzadC for 72 h; ( B ) RT–PCR after treatment of CML-T1 and EM-2 with 3 µM AzadC for 24, 48 and 72 h; ( C ) immunoblotting after treatment of CML-T1 and LAMA-84 with 1 and 3 µM AzadC for 72 h; ( D ) RT–PCR after treatment of BV-173 for 24, 48 and 72 h and RPMI-8226 for 72 h with 3 µM AzadC.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation

Restriction-PCR-assay of the IRF-4 promoter in hematopoietic cells. ( A ) Simplified structure of the CpG sites in the human IRF-4 promoter region including exon 1. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream). Restriction sites, fragments (F1, F2) of the restriction-PCR-assay and regions amplified after bisulfite treatment for sequencing are shown below. The numbers in italics refer to the sequence data by Grossman et al . ( 30 ). ( B and C ) Restriction-PCR-assay. Restriction of DNA with EcoRI (E, no recognition site), HpaII (H, sensitive), Bsp143II (BII, sensitive), Bsh1236I (BI, sensitive) and MspI (M, resistant) and subsequent PCR amplification. Three representative IRF-4-negative (K-562, CML-T1, EM-2) and two IRF-4-positive cell lines (SD-1, BV-173) are shown. IRF-4 expression is denoted on the right. ( B ) PCR-fragment 1 (F1); ( C ) PCR-fragment 2 (F2).
Figure Legend Snippet: Restriction-PCR-assay of the IRF-4 promoter in hematopoietic cells. ( A ) Simplified structure of the CpG sites in the human IRF-4 promoter region including exon 1. Each CpG motif is shown as circle and numbered (above, labeling beginning from the 3′ end moving upstream). Restriction sites, fragments (F1, F2) of the restriction-PCR-assay and regions amplified after bisulfite treatment for sequencing are shown below. The numbers in italics refer to the sequence data by Grossman et al . ( 30 ). ( B and C ) Restriction-PCR-assay. Restriction of DNA with EcoRI (E, no recognition site), HpaII (H, sensitive), Bsp143II (BII, sensitive), Bsh1236I (BI, sensitive) and MspI (M, resistant) and subsequent PCR amplification. Three representative IRF-4-negative (K-562, CML-T1, EM-2) and two IRF-4-positive cell lines (SD-1, BV-173) are shown. IRF-4 expression is denoted on the right. ( B ) PCR-fragment 1 (F1); ( C ) PCR-fragment 2 (F2).

Techniques Used: Polymerase Chain Reaction, Labeling, Amplification, Sequencing, Expressing

Correlation of IRF-4 mRNA expression and nucleotide changes in the IRF-4 promoter. ( A ) RT–PCR of different hematopoietic cell lines. ( B ) nucleotide changes in the promoter of IRF-4-positive or -negative cells in comparison to the germline sequence at positions 98, 111 and 1063 (GenBank accession no. U52683 ), representing nucleotide −1081, −1068 and −116 ( 30 ) or −1094, −1081 and −129 ( 31 ), respectively.
Figure Legend Snippet: Correlation of IRF-4 mRNA expression and nucleotide changes in the IRF-4 promoter. ( A ) RT–PCR of different hematopoietic cell lines. ( B ) nucleotide changes in the promoter of IRF-4-positive or -negative cells in comparison to the germline sequence at positions 98, 111 and 1063 (GenBank accession no. U52683 ), representing nucleotide −1081, −1068 and −116 ( 30 ) or −1094, −1081 and −129 ( 31 ), respectively.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing

45) Product Images from "Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana"

Article Title: Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana

Journal: Plant Methods

doi: 10.1186/s13007-018-0343-2

Effects of co‐expressing virus‐derived genes encoding cell cycle regulatory proteins. Agrobacteria strain AGL1 harbouring pEAQ‐GSN were co‐infiltrated into N. benthamiana leaves with a TYDV Rep / RepA or RepA genes and the TYDV RepA gene containing a LxCxK mutation in the RB-binding motif, and b cell cycle regulatory genes from related circular ssDNA plant viruses, under the transcriptional control of the truncated CaMV 35S (− 90) promoter (Δ35S). Leaves were sampled 4 dpi and TSP extracted for GUS fluorometric enzyme assays. Columns represent relative levels of mean GUS enzyme activities and bars represent ± SE. (†) indicates the reference treatment and (*) indicates data significantly different to the reference ( p
Figure Legend Snippet: Effects of co‐expressing virus‐derived genes encoding cell cycle regulatory proteins. Agrobacteria strain AGL1 harbouring pEAQ‐GSN were co‐infiltrated into N. benthamiana leaves with a TYDV Rep / RepA or RepA genes and the TYDV RepA gene containing a LxCxK mutation in the RB-binding motif, and b cell cycle regulatory genes from related circular ssDNA plant viruses, under the transcriptional control of the truncated CaMV 35S (− 90) promoter (Δ35S). Leaves were sampled 4 dpi and TSP extracted for GUS fluorometric enzyme assays. Columns represent relative levels of mean GUS enzyme activities and bars represent ± SE. (†) indicates the reference treatment and (*) indicates data significantly different to the reference ( p

Techniques Used: Expressing, Derivative Assay, Mutagenesis, Binding Assay

Schematic representation of pEAQ-GSN, pSPECIAL and pNEEDS vectors. pEAQ-GSN was assembled by introducing the uid A reporter gene and small synthetic intron (syntron) between the CPMV 5′ and 3′ UTR translation enhancers in pEAQ-HT [ 31 ]. pSPECIAL is based on pEAQ-GSN with a downstream expression cassette encoding the truncated CMV 2b (1-94) silencing suppressor protein. pNEEDS is a pBIN-Plus binary vector comprising two expression cassettes encoding the AtBAG 4 stress tolerance protein under the transcriptional control of the nos promoter and the TYDV Rep/RepA cell cycle control gene products under the transcriptional control of the truncated CaMV (− 90) promoter. 35SP = Cauliflower mosaic virus 35S promoter; CPMV 5′ UTR = Cowpea mosaic virus RNA‐2 5′UTR; uid A = gene encoding GUS; syntron = synthetic intron; CPMV 3′ UTR = Cowpea mosaic virus RNA‐2 3′UTR; nosT = nopaline synthase terminator from Agrobacterium ; TBSV p19 = Tomato bushy stunt virus p19 silencing suppressor gene; 35ST = Cauliflower mosaic virus 35S terminator; CMV 2b (1-94) = Cucumber mosaic virus truncated 2b silencing suppressor gene (amino acids 1-94); nosP = nopaline synthase promoter from Agrobacterium ; AtBAG 4= Arabidopsis BAG 4 gene; ∆35SP = truncated Cauliflower mosaic virus 35S (− 90) promoter; TYDV Rep / RepA = Tobacco yellow dwarf virus Rep / RepA gene encoding both Rep and RepA
Figure Legend Snippet: Schematic representation of pEAQ-GSN, pSPECIAL and pNEEDS vectors. pEAQ-GSN was assembled by introducing the uid A reporter gene and small synthetic intron (syntron) between the CPMV 5′ and 3′ UTR translation enhancers in pEAQ-HT [ 31 ]. pSPECIAL is based on pEAQ-GSN with a downstream expression cassette encoding the truncated CMV 2b (1-94) silencing suppressor protein. pNEEDS is a pBIN-Plus binary vector comprising two expression cassettes encoding the AtBAG 4 stress tolerance protein under the transcriptional control of the nos promoter and the TYDV Rep/RepA cell cycle control gene products under the transcriptional control of the truncated CaMV (− 90) promoter. 35SP = Cauliflower mosaic virus 35S promoter; CPMV 5′ UTR = Cowpea mosaic virus RNA‐2 5′UTR; uid A = gene encoding GUS; syntron = synthetic intron; CPMV 3′ UTR = Cowpea mosaic virus RNA‐2 3′UTR; nosT = nopaline synthase terminator from Agrobacterium ; TBSV p19 = Tomato bushy stunt virus p19 silencing suppressor gene; 35ST = Cauliflower mosaic virus 35S terminator; CMV 2b (1-94) = Cucumber mosaic virus truncated 2b silencing suppressor gene (amino acids 1-94); nosP = nopaline synthase promoter from Agrobacterium ; AtBAG 4= Arabidopsis BAG 4 gene; ∆35SP = truncated Cauliflower mosaic virus 35S (− 90) promoter; TYDV Rep / RepA = Tobacco yellow dwarf virus Rep / RepA gene encoding both Rep and RepA

Techniques Used: Expressing, Plasmid Preparation

46) Product Images from "Helicobacter Pylori and Toll-Like Receptor Agonists Induce Syndecan-4 Expression in an NF-?B-Dependent Manner"

Article Title: Helicobacter Pylori and Toll-Like Receptor Agonists Induce Syndecan-4 Expression in an NF-?B-Dependent Manner

Journal: Glycobiology

doi: 10.1093/glycob/cwj061

H. pylori- induced syndecan-4 promoter activity is NF-κB dependent
Figure Legend Snippet: H. pylori- induced syndecan-4 promoter activity is NF-κB dependent

Techniques Used: Activity Assay

Mutation of the syndecan-4 NF-κB-binding site inhibits inducible promoter activity
Figure Legend Snippet: Mutation of the syndecan-4 NF-κB-binding site inhibits inducible promoter activity

Techniques Used: Mutagenesis, Binding Assay, Activity Assay

Inducible cell surface expression of syndecan-4 on MKN45 cells
Figure Legend Snippet: Inducible cell surface expression of syndecan-4 on MKN45 cells

Techniques Used: Expressing

LPS-induced expression of syndecan-4 in RAW264.7 macrophages
Figure Legend Snippet: LPS-induced expression of syndecan-4 in RAW264.7 macrophages

Techniques Used: Expressing

Inhibition of syndecan-4 mRNA expression by MG-132
Figure Legend Snippet: Inhibition of syndecan-4 mRNA expression by MG-132

Techniques Used: Inhibition, Expressing

Electrophoretic mobility shift assays (EMSA) of an NF-κB-binding site in the human syndecan-4 promoter
Figure Legend Snippet: Electrophoretic mobility shift assays (EMSA) of an NF-κB-binding site in the human syndecan-4 promoter

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay

Regulation of syndecan-4 mRNA expression in gastric epithelial cells
Figure Legend Snippet: Regulation of syndecan-4 mRNA expression in gastric epithelial cells

Techniques Used: Expressing

47) Product Images from "E2A and IRF-4/Pip Promote Chromatin Modification and Transcription of the Immunoglobulin ? Locus in Pre-B Cells"

Article Title: E2A and IRF-4/Pip Promote Chromatin Modification and Transcription of the Immunoglobulin ? Locus in Pre-B Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.3.810-821.2006

IRF-4 expression promotes E2A association with κE3′ in pre-B cells. (A) Increased E2A association with the Igκ enhancers upon locus activation. A pre-B-cell line carrying a dual-affinity-tagged E2A ( E2A FH ) was treated with or without 10 μM STI-571 for 6 h. Relative E2A association with the Igκ intronic (κEi) and 3′ (κE3′) enhancers was determined by quantitative PCR. E2A enrichment at the Ig heavy chain (IgH) intronic enhancer (Eμ) and CD19 promoter serve as positive and negative controls for E2A DNA binding, respectively. Results are presented as ratio of IP to input DNA and are the averages of two independent IP ± standard deviations. (B) IRF-4 and E2A gene expression in E2A FH and E2A FH IRF-4 KD pre-B cells with or without STI treatment. Total RNA was prepared from a portion of the cells used as described for panel A, and the relative expression levels of IRF-4 and E2A mRNA were determined by quantitative RT-PCR. IRF-4 and E2A transcript levels were normalized to the housekeeping gene EF1α. (C) IRF-4 expression influences E2A association with κE3′ and Ig heavy chain Eμ. Dual-affinity-tagged E2A was immunoprecipitated from E2A FH/FH IRF-4 KD pre-B-cell lines treated for 6 h with or without 10 μM STI-571. Relative E2A associations with κE3′, Ig heavy chain Eμ, and the mb-1 promoter were determined by quantitative PCR and are presented as ratio of IP to input DNA. E2A enrichment at the CD19 promoter is a negative control.
Figure Legend Snippet: IRF-4 expression promotes E2A association with κE3′ in pre-B cells. (A) Increased E2A association with the Igκ enhancers upon locus activation. A pre-B-cell line carrying a dual-affinity-tagged E2A ( E2A FH ) was treated with or without 10 μM STI-571 for 6 h. Relative E2A association with the Igκ intronic (κEi) and 3′ (κE3′) enhancers was determined by quantitative PCR. E2A enrichment at the Ig heavy chain (IgH) intronic enhancer (Eμ) and CD19 promoter serve as positive and negative controls for E2A DNA binding, respectively. Results are presented as ratio of IP to input DNA and are the averages of two independent IP ± standard deviations. (B) IRF-4 and E2A gene expression in E2A FH and E2A FH IRF-4 KD pre-B cells with or without STI treatment. Total RNA was prepared from a portion of the cells used as described for panel A, and the relative expression levels of IRF-4 and E2A mRNA were determined by quantitative RT-PCR. IRF-4 and E2A transcript levels were normalized to the housekeeping gene EF1α. (C) IRF-4 expression influences E2A association with κE3′ and Ig heavy chain Eμ. Dual-affinity-tagged E2A was immunoprecipitated from E2A FH/FH IRF-4 KD pre-B-cell lines treated for 6 h with or without 10 μM STI-571. Relative E2A associations with κE3′, Ig heavy chain Eμ, and the mb-1 promoter were determined by quantitative PCR and are presented as ratio of IP to input DNA. E2A enrichment at the CD19 promoter is a negative control.

Techniques Used: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Binding Assay, Quantitative RT-PCR, Immunoprecipitation, Negative Control

Igκ germ line transcription and recombination is dependent on E2A. (A) Schematic of the 3′ region of the murine Igκ locus, highlighting the germ line transcript promoters, intronic enhancer, and 3′ enhancer. (B) E2A and Igκ expression in E2A-deficient Ab-MuLV pre-B cells. Primers for murine E47 (E2A) and Igκ constant exon (Igκ-C) were used to evaluate expression of these genes in E2A +/+ cells and three independent E2A −/− clones. EF1α serves as a positive loading control. (C) E2A-deficient Ab-MuLV pre-B cells fail to initiate light chain recombination. E2A +/+ or E2A −/− cells untreated (C) or treated with LPS or STI-571 were assayed for the production of RAG-mediated Jκ1 or Jλ signal break ends (SBE) by LM-PCR. CD14 gene amplification serves as a positive control for DNA loading and quality. (D) Expression of transcripts involved in Igκ gene regulation in E2A-deficient pre-B cells. E2A +/+ and E2A −/− cells were incubated for 5 h at 37°C with or without 10 μM STI-571, and the relative abundance of various transcripts was measured by semiquantitative RT-PCR. The expression levels of Igκ germ line transcript (Igκ-GL), RAG1, RAG2, and transcription factors known to regulate Igκ expression were examined. Each sample is presented as a series of threefold serial dilutions. EF1α was used to verify equivalent cDNA loading and quality between samples.
Figure Legend Snippet: Igκ germ line transcription and recombination is dependent on E2A. (A) Schematic of the 3′ region of the murine Igκ locus, highlighting the germ line transcript promoters, intronic enhancer, and 3′ enhancer. (B) E2A and Igκ expression in E2A-deficient Ab-MuLV pre-B cells. Primers for murine E47 (E2A) and Igκ constant exon (Igκ-C) were used to evaluate expression of these genes in E2A +/+ cells and three independent E2A −/− clones. EF1α serves as a positive loading control. (C) E2A-deficient Ab-MuLV pre-B cells fail to initiate light chain recombination. E2A +/+ or E2A −/− cells untreated (C) or treated with LPS or STI-571 were assayed for the production of RAG-mediated Jκ1 or Jλ signal break ends (SBE) by LM-PCR. CD14 gene amplification serves as a positive control for DNA loading and quality. (D) Expression of transcripts involved in Igκ gene regulation in E2A-deficient pre-B cells. E2A +/+ and E2A −/− cells were incubated for 5 h at 37°C with or without 10 μM STI-571, and the relative abundance of various transcripts was measured by semiquantitative RT-PCR. The expression levels of Igκ germ line transcript (Igκ-GL), RAG1, RAG2, and transcription factors known to regulate Igκ expression were examined. Each sample is presented as a series of threefold serial dilutions. EF1α was used to verify equivalent cDNA loading and quality between samples.

Techniques Used: Expressing, Clone Assay, Polymerase Chain Reaction, Amplification, Positive Control, Incubation, Reverse Transcription Polymerase Chain Reaction

E2A gene status influences Igκ enhancer histone acetylation. (A) Chromatin modification of the Igκ enhancers in E2A −/− pre-B cells. Mononucleosomes were prepared from E2A +/+ pre-B-cell lines treated for 5 h with or without 10 μM STI-571, and acetylated H3 histones were immunoprecipitated. Relative enrichment of H3 acetylation sequences was determined by quantitative PCR and plotted as IP DNA over input DNA. Enrichment of sequences corresponding to the Igκ intronic (κEi) and 3′ (κE3′) core enhancers was determined. Enrichment of the housekeeping gene G6PD and the silent trypsinogen locus T4D served as positive and negative internal controls, respectively. Two independently derived E2A −/− clones treated with STI-571 were analyzed. (B) Mononucleosomes prepared from the pre-B cells described for panel A were immunoprecipitated and analyzed for H4 acetylated histones at various loci. Relative enrichment of acetylated H4 was plotted as IP/input. Acetyl-H4 enrichment at the G6PD and T4D loci and the Igκ enhancers (κEi and κE3′) was determined by quantitative PCR. Two independently derived E2A −/− clones treated with STI-571 were analyzed.
Figure Legend Snippet: E2A gene status influences Igκ enhancer histone acetylation. (A) Chromatin modification of the Igκ enhancers in E2A −/− pre-B cells. Mononucleosomes were prepared from E2A +/+ pre-B-cell lines treated for 5 h with or without 10 μM STI-571, and acetylated H3 histones were immunoprecipitated. Relative enrichment of H3 acetylation sequences was determined by quantitative PCR and plotted as IP DNA over input DNA. Enrichment of sequences corresponding to the Igκ intronic (κEi) and 3′ (κE3′) core enhancers was determined. Enrichment of the housekeeping gene G6PD and the silent trypsinogen locus T4D served as positive and negative internal controls, respectively. Two independently derived E2A −/− clones treated with STI-571 were analyzed. (B) Mononucleosomes prepared from the pre-B cells described for panel A were immunoprecipitated and analyzed for H4 acetylated histones at various loci. Relative enrichment of acetylated H4 was plotted as IP/input. Acetyl-H4 enrichment at the G6PD and T4D loci and the Igκ enhancers (κEi and κE3′) was determined by quantitative PCR. Two independently derived E2A −/− clones treated with STI-571 were analyzed.

Techniques Used: Modification, Immunoprecipitation, Real-time Polymerase Chain Reaction, Derivative Assay, Clone Assay

E47R promotes histone acetylation of the Igκ enhancers and germ line promoters. Mononucleosomes were prepared from E47R-reconstituted pre-B cells treated with 0.1% DMSO or 1 μM tamoxifen for 24 h. Enrichment of acetylated histone H3 or acetylated histone H4 at the 5′ κ o and Jκ1 proximal Igκ germ line promoters and the intronic (κEi) and 3′ (κE3′) enhancers was examined. Relative acetylation was determined by quantitative PCR and plotted as IP/input. The active G6PD and silent T4D loci serve as positive and negative controls, respectively.
Figure Legend Snippet: E47R promotes histone acetylation of the Igκ enhancers and germ line promoters. Mononucleosomes were prepared from E47R-reconstituted pre-B cells treated with 0.1% DMSO or 1 μM tamoxifen for 24 h. Enrichment of acetylated histone H3 or acetylated histone H4 at the 5′ κ o and Jκ1 proximal Igκ germ line promoters and the intronic (κEi) and 3′ (κE3′) enhancers was examined. Relative acetylation was determined by quantitative PCR and plotted as IP/input. The active G6PD and silent T4D loci serve as positive and negative controls, respectively.

Techniques Used: Real-time Polymerase Chain Reaction

48) Product Images from "Human Natural Killer T Cells Infiltrate into the Skin at Elicitation Sites of Allergic Contact Dermatitis"

Article Title: Human Natural Killer T Cells Infiltrate into the Skin at Elicitation Sites of Allergic Contact Dermatitis

Journal: The Journal of investigative dermatology

doi: 10.1038/sj.jid.5701199

Unique Vα24JαQ NKT-associated TCR gene expression occurs in ACD, but not in normal skin Nested PCR to detect unique Vα24-JαQ TCR sequences in skin biopsy specimens. RNA was extracted from an NKT cell line, Jurkat cells, and foreskin KC (FSKC), cDNA was synthesized, subjected to the nested PCR, and run on an agarose gel as described in Methods. ( a ) Control lane (Ctrl) represents no cDNA added to nested PCR. ( b ) This same nested PCR was applied to normal skin (NORM), psoriasis (PSOR) and five cases of ACD (CD1–5). ( c ) Nested PCR from same specimens was subjected to real-time PCR amplification, with SYBR green as the fluorochrome (* P
Figure Legend Snippet: Unique Vα24JαQ NKT-associated TCR gene expression occurs in ACD, but not in normal skin Nested PCR to detect unique Vα24-JαQ TCR sequences in skin biopsy specimens. RNA was extracted from an NKT cell line, Jurkat cells, and foreskin KC (FSKC), cDNA was synthesized, subjected to the nested PCR, and run on an agarose gel as described in Methods. ( a ) Control lane (Ctrl) represents no cDNA added to nested PCR. ( b ) This same nested PCR was applied to normal skin (NORM), psoriasis (PSOR) and five cases of ACD (CD1–5). ( c ) Nested PCR from same specimens was subjected to real-time PCR amplification, with SYBR green as the fluorochrome (* P

Techniques Used: Expressing, Nested PCR, Synthesized, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, SYBR Green Assay

Infiltrating NKT cells in ACD express both transcripts for the cytokines IFN-γ and IL-4 ACD skin-biopsy specimens studied by ISH: double-labeled with antisense oligoprobes for Vα24 (left panel, green; middle panel, cytokine ( a ) (IFN-γ) or ( b ) IL-4 (red); right panel, overlays) (bar=10 μm).
Figure Legend Snippet: Infiltrating NKT cells in ACD express both transcripts for the cytokines IFN-γ and IL-4 ACD skin-biopsy specimens studied by ISH: double-labeled with antisense oligoprobes for Vα24 (left panel, green; middle panel, cytokine ( a ) (IFN-γ) or ( b ) IL-4 (red); right panel, overlays) (bar=10 μm).

Techniques Used: In Situ Hybridization, Labeling

CD1d+ Monocytic APCs, but not CD1d+ KC, induce cytokine gene expression and secretion by NKT-cells in vitro A polyclonal NKT cell line was cultured with the CD1d+ cell line, THP-1 or CD1d+ KC (differentiated or undifferentiated, and treated with IFN-γ, see Methods) in the presence ( a and b ) or absence (data not shown) of α-galactosylceramide for 24 hours, and supernatants were harvested for specific luminex assay. ( a ) IFN-γ; ( b ) IL-4. In another experiment, the NKT cells were isolated after 6 hours of incubation with the above cell lines, and RNA was extracted, cDNA was synthesized, and gene expression was studied by ( c ) real-time PCR IFN-γ; ( d ) IL-4. (* P
Figure Legend Snippet: CD1d+ Monocytic APCs, but not CD1d+ KC, induce cytokine gene expression and secretion by NKT-cells in vitro A polyclonal NKT cell line was cultured with the CD1d+ cell line, THP-1 or CD1d+ KC (differentiated or undifferentiated, and treated with IFN-γ, see Methods) in the presence ( a and b ) or absence (data not shown) of α-galactosylceramide for 24 hours, and supernatants were harvested for specific luminex assay. ( a ) IFN-γ; ( b ) IL-4. In another experiment, the NKT cells were isolated after 6 hours of incubation with the above cell lines, and RNA was extracted, cDNA was synthesized, and gene expression was studied by ( c ) real-time PCR IFN-γ; ( d ) IL-4. (* P

Techniques Used: Expressing, In Vitro, Cell Culture, Luminex, Isolation, Incubation, Synthesized, Real-time Polymerase Chain Reaction

49) Product Images from "Human Natural Killer T Cells Infiltrate into the Skin at Elicitation Sites of Allergic Contact Dermatitis"

Article Title: Human Natural Killer T Cells Infiltrate into the Skin at Elicitation Sites of Allergic Contact Dermatitis

Journal: The Journal of investigative dermatology

doi: 10.1038/sj.jid.5701199

Unique Vα24JαQ NKT-associated TCR gene expression occurs in ACD, but not in normal skin Nested PCR to detect unique Vα24-JαQ TCR sequences in skin biopsy specimens. RNA was extracted from an NKT cell line, Jurkat cells, and foreskin KC (FSKC), cDNA was synthesized, subjected to the nested PCR, and run on an agarose gel as described in Methods. ( a ) Control lane (Ctrl) represents no cDNA added to nested PCR. ( b ) This same nested PCR was applied to normal skin (NORM), psoriasis (PSOR) and five cases of ACD (CD1–5). ( c ) Nested PCR from same specimens was subjected to real-time PCR amplification, with SYBR green as the fluorochrome (* P
Figure Legend Snippet: Unique Vα24JαQ NKT-associated TCR gene expression occurs in ACD, but not in normal skin Nested PCR to detect unique Vα24-JαQ TCR sequences in skin biopsy specimens. RNA was extracted from an NKT cell line, Jurkat cells, and foreskin KC (FSKC), cDNA was synthesized, subjected to the nested PCR, and run on an agarose gel as described in Methods. ( a ) Control lane (Ctrl) represents no cDNA added to nested PCR. ( b ) This same nested PCR was applied to normal skin (NORM), psoriasis (PSOR) and five cases of ACD (CD1–5). ( c ) Nested PCR from same specimens was subjected to real-time PCR amplification, with SYBR green as the fluorochrome (* P

Techniques Used: Expressing, Nested PCR, Synthesized, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Amplification, SYBR Green Assay

Infiltrating NKT cells in ACD express both transcripts for the cytokines IFN-γ and IL-4 ACD skin-biopsy specimens studied by ISH: double-labeled with antisense oligoprobes for Vα24 (left panel, green; middle panel, cytokine ( a ) (IFN-γ) or ( b ) IL-4 (red); right panel, overlays) (bar=10 μm).
Figure Legend Snippet: Infiltrating NKT cells in ACD express both transcripts for the cytokines IFN-γ and IL-4 ACD skin-biopsy specimens studied by ISH: double-labeled with antisense oligoprobes for Vα24 (left panel, green; middle panel, cytokine ( a ) (IFN-γ) or ( b ) IL-4 (red); right panel, overlays) (bar=10 μm).

Techniques Used: In Situ Hybridization, Labeling

CD3 gene expression is more robust than Vα24 expression in ACD skin biopsy specimens Real-time PCR was utilized to detect ( a ) CD3 ε gene expression (a molecular marker for all TCR-bearing T cells) or ( b ) Vα24 (a molecular marker for NKT cells) using identical methods (single round of PCR amplification, with SYBER green fluorescence dye for real-time PCR detection. (* P
Figure Legend Snippet: CD3 gene expression is more robust than Vα24 expression in ACD skin biopsy specimens Real-time PCR was utilized to detect ( a ) CD3 ε gene expression (a molecular marker for all TCR-bearing T cells) or ( b ) Vα24 (a molecular marker for NKT cells) using identical methods (single round of PCR amplification, with SYBER green fluorescence dye for real-time PCR detection. (* P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Marker, Polymerase Chain Reaction, Amplification, Fluorescence

CD1d+ Monocytic APCs, but not CD1d+ KC, induce cytokine gene expression and secretion by NKT-cells in vitro A polyclonal NKT cell line was cultured with the CD1d+ cell line, THP-1 or CD1d+ KC (differentiated or undifferentiated, and treated with IFN-γ, see Methods) in the presence ( a and b ) or absence (data not shown) of α-galactosylceramide for 24 hours, and supernatants were harvested for specific luminex assay. ( a ) IFN-γ; ( b ) IL-4. In another experiment, the NKT cells were isolated after 6 hours of incubation with the above cell lines, and RNA was extracted, cDNA was synthesized, and gene expression was studied by ( c ) real-time PCR IFN-γ; ( d ) IL-4. (* P
Figure Legend Snippet: CD1d+ Monocytic APCs, but not CD1d+ KC, induce cytokine gene expression and secretion by NKT-cells in vitro A polyclonal NKT cell line was cultured with the CD1d+ cell line, THP-1 or CD1d+ KC (differentiated or undifferentiated, and treated with IFN-γ, see Methods) in the presence ( a and b ) or absence (data not shown) of α-galactosylceramide for 24 hours, and supernatants were harvested for specific luminex assay. ( a ) IFN-γ; ( b ) IL-4. In another experiment, the NKT cells were isolated after 6 hours of incubation with the above cell lines, and RNA was extracted, cDNA was synthesized, and gene expression was studied by ( c ) real-time PCR IFN-γ; ( d ) IL-4. (* P

Techniques Used: Expressing, In Vitro, Cell Culture, Luminex, Isolation, Incubation, Synthesized, Real-time Polymerase Chain Reaction

50) Product Images from "Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression"

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression

Journal: BMC Immunology

doi: 10.1186/s12865-017-0198-8

Detection of cell surface molecules and signaling pathway molecules after knockdown and over-expression of Foxo1 in Treg cells. a , Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with Foxo1 siRNA by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 siRNA). b Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with over-expression plasmid of Foxo1 by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 over-expression). c Detected of Median Fluorescence Intensity (MFI) for CD127 in Treg cells 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid by flow cytometry. d Representative western blot of p-Erk1/2, total Erk1/2, p-Akt, total Akt, p-Stat5, total Stat5, p-Foxo1 and total Foxo1 in Treg cells 48 h after transfection with Foxo1 siRNA and over-expression plasmid of Foxo1, GADPH was used as a control. Treg cells stimulated with anti-CD3 (0.01 μg/ml) and anti-CD28 (1.0 μg/ml) in medium during culture. e Expression of mRNA for IL-2, IL-4, IL-7 and IL-15 in Treg cells, 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid. All experiments were repeated at least three times. ** P
Figure Legend Snippet: Detection of cell surface molecules and signaling pathway molecules after knockdown and over-expression of Foxo1 in Treg cells. a , Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with Foxo1 siRNA by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 siRNA). b Representative expression of the Foxo1, CD127, CD103, ICOS, Foxp3 or CD25 in Treg cells 48 h after transfection with over-expression plasmid of Foxo1 by flow cytometry (broken black line: isotype, green line: control, red line: Foxo1 over-expression). c Detected of Median Fluorescence Intensity (MFI) for CD127 in Treg cells 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid by flow cytometry. d Representative western blot of p-Erk1/2, total Erk1/2, p-Akt, total Akt, p-Stat5, total Stat5, p-Foxo1 and total Foxo1 in Treg cells 48 h after transfection with Foxo1 siRNA and over-expression plasmid of Foxo1, GADPH was used as a control. Treg cells stimulated with anti-CD3 (0.01 μg/ml) and anti-CD28 (1.0 μg/ml) in medium during culture. e Expression of mRNA for IL-2, IL-4, IL-7 and IL-15 in Treg cells, 48 h after transfection with Foxo1 siRNA and Foxo1 over-expression plasmid. All experiments were repeated at least three times. ** P

Techniques Used: Over Expression, Expressing, Transfection, Flow Cytometry, Cytometry, Plasmid Preparation, Fluorescence, Western Blot

51) Product Images from "Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells"

Article Title: Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144397

Growth kinetics of E . coli ghosts harbouring pET28a- E lysis plasmid. The transformed bacteria with gene E of bacteriophage PhiX174 was grown at 37°C until mid-log phase (OD600 = 0.25). The culture was split and one part was induced by the addition of 1 mM IPTG whereas the other part served as a non-induced control. The growth was monitored by measuring the optical density (OD 600nm ) of culture at various time points. After induction (indicated by arrow), BGs showed rapid decrease in OD indicative of lysis whereas the uninduced culture showed growth of bacteria as indicated by an increase in OD.
Figure Legend Snippet: Growth kinetics of E . coli ghosts harbouring pET28a- E lysis plasmid. The transformed bacteria with gene E of bacteriophage PhiX174 was grown at 37°C until mid-log phase (OD600 = 0.25). The culture was split and one part was induced by the addition of 1 mM IPTG whereas the other part served as a non-induced control. The growth was monitored by measuring the optical density (OD 600nm ) of culture at various time points. After induction (indicated by arrow), BGs showed rapid decrease in OD indicative of lysis whereas the uninduced culture showed growth of bacteria as indicated by an increase in OD.

Techniques Used: Lysis, Plasmid Preparation, Transformation Assay

52) Product Images from "E2A and IRF-4/Pip Promote Chromatin Modification and Transcription of the Immunoglobulin ? Locus in Pre-B Cells"

Article Title: E2A and IRF-4/Pip Promote Chromatin Modification and Transcription of the Immunoglobulin ? Locus in Pre-B Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.3.810-821.2006

Inducible human E47 rescues Igκ germ line transcription and recombination. (A) E47R is sufficient to activate Igκ germ line transcription. Total RNA was isolated from E2A +/+ or E2A −/− Ab-MuLV pre-B cells treated with 1 μM tamoxifen (tamox) and E47R-reconstituted Ab-MuLV B cells treated with 0.1% DMSO or 1 μM tamoxifen for 6 h. Semiquantitative RT-PCR was performed to determine the relative level of Igκ germ line transcription (Igκ-GL). EF1α serves as a positive loading control, and results are presented as threefold serial dilutions. (B) E47R promotes Igκ recombination. E47R-reconstituted pre-B cells were treated with 1 μM tamoxifen or 0.1% DMSO for 8 or 24 h. Genomic DNA was prepared from the cells, and the accumulation of Jκ1 signal break ends (SBE) was determined by LM-PCR. PCR amplification of the CD14 gene serves as a control for DNA loading and quality. (C) Igκ germ line transcription is dependent on E2A. E47R-reconstituted pre-B cells were treated with 1 μM tamoxifen for 6 h, washed, and cultured without tamoxifen. Total RNA was prepared at the indicated times after the removal of tamoxifen. Mock-treated cells were cultured in the presence of 0.1% DMSO for the entire duration of the experiment. Relative abundance of Igκ germ line transcript and EF1α transcript was determined by RT-PCR.
Figure Legend Snippet: Inducible human E47 rescues Igκ germ line transcription and recombination. (A) E47R is sufficient to activate Igκ germ line transcription. Total RNA was isolated from E2A +/+ or E2A −/− Ab-MuLV pre-B cells treated with 1 μM tamoxifen (tamox) and E47R-reconstituted Ab-MuLV B cells treated with 0.1% DMSO or 1 μM tamoxifen for 6 h. Semiquantitative RT-PCR was performed to determine the relative level of Igκ germ line transcription (Igκ-GL). EF1α serves as a positive loading control, and results are presented as threefold serial dilutions. (B) E47R promotes Igκ recombination. E47R-reconstituted pre-B cells were treated with 1 μM tamoxifen or 0.1% DMSO for 8 or 24 h. Genomic DNA was prepared from the cells, and the accumulation of Jκ1 signal break ends (SBE) was determined by LM-PCR. PCR amplification of the CD14 gene serves as a control for DNA loading and quality. (C) Igκ germ line transcription is dependent on E2A. E47R-reconstituted pre-B cells were treated with 1 μM tamoxifen for 6 h, washed, and cultured without tamoxifen. Total RNA was prepared at the indicated times after the removal of tamoxifen. Mock-treated cells were cultured in the presence of 0.1% DMSO for the entire duration of the experiment. Relative abundance of Igκ germ line transcript and EF1α transcript was determined by RT-PCR.

Techniques Used: Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Cell Culture

Igκ germ line transcription and recombination is dependent on E2A. (A) Schematic of the 3′ region of the murine Igκ locus, highlighting the germ line transcript promoters, intronic enhancer, and 3′ enhancer. (B) E2A and Igκ expression in E2A-deficient Ab-MuLV pre-B cells. Primers for murine E47 (E2A) and Igκ constant exon (Igκ-C) were used to evaluate expression of these genes in E2A +/+ cells and three independent E2A −/− clones. EF1α serves as a positive loading control. (C) E2A-deficient Ab-MuLV pre-B cells fail to initiate light chain recombination. E2A +/+ or E2A −/− cells untreated (C) or treated with LPS or STI-571 were assayed for the production of RAG-mediated Jκ1 or Jλ signal break ends (SBE) by LM-PCR. CD14 gene amplification serves as a positive control for DNA loading and quality. (D) Expression of transcripts involved in Igκ gene regulation in E2A-deficient pre-B cells. E2A +/+ and E2A −/− cells were incubated for 5 h at 37°C with or without 10 μM STI-571, and the relative abundance of various transcripts was measured by semiquantitative RT-PCR. The expression levels of Igκ germ line transcript (Igκ-GL), RAG1, RAG2, and transcription factors known to regulate Igκ expression were examined. Each sample is presented as a series of threefold serial dilutions. EF1α was used to verify equivalent cDNA loading and quality between samples.
Figure Legend Snippet: Igκ germ line transcription and recombination is dependent on E2A. (A) Schematic of the 3′ region of the murine Igκ locus, highlighting the germ line transcript promoters, intronic enhancer, and 3′ enhancer. (B) E2A and Igκ expression in E2A-deficient Ab-MuLV pre-B cells. Primers for murine E47 (E2A) and Igκ constant exon (Igκ-C) were used to evaluate expression of these genes in E2A +/+ cells and three independent E2A −/− clones. EF1α serves as a positive loading control. (C) E2A-deficient Ab-MuLV pre-B cells fail to initiate light chain recombination. E2A +/+ or E2A −/− cells untreated (C) or treated with LPS or STI-571 were assayed for the production of RAG-mediated Jκ1 or Jλ signal break ends (SBE) by LM-PCR. CD14 gene amplification serves as a positive control for DNA loading and quality. (D) Expression of transcripts involved in Igκ gene regulation in E2A-deficient pre-B cells. E2A +/+ and E2A −/− cells were incubated for 5 h at 37°C with or without 10 μM STI-571, and the relative abundance of various transcripts was measured by semiquantitative RT-PCR. The expression levels of Igκ germ line transcript (Igκ-GL), RAG1, RAG2, and transcription factors known to regulate Igκ expression were examined. Each sample is presented as a series of threefold serial dilutions. EF1α was used to verify equivalent cDNA loading and quality between samples.

Techniques Used: Expressing, Clone Assay, Polymerase Chain Reaction, Amplification, Positive Control, Incubation, Reverse Transcription Polymerase Chain Reaction

E47R promotes histone acetylation of the Igκ enhancers and germ line promoters. Mononucleosomes were prepared from E47R-reconstituted pre-B cells treated with 0.1% DMSO or 1 μM tamoxifen for 24 h. Enrichment of acetylated histone H3 or acetylated histone H4 at the 5′ κ o and Jκ1 proximal Igκ germ line promoters and the intronic (κEi) and 3′ (κE3′) enhancers was examined. Relative acetylation was determined by quantitative PCR and plotted as IP/input. The active G6PD and silent T4D loci serve as positive and negative controls, respectively.
Figure Legend Snippet: E47R promotes histone acetylation of the Igκ enhancers and germ line promoters. Mononucleosomes were prepared from E47R-reconstituted pre-B cells treated with 0.1% DMSO or 1 μM tamoxifen for 24 h. Enrichment of acetylated histone H3 or acetylated histone H4 at the 5′ κ o and Jκ1 proximal Igκ germ line promoters and the intronic (κEi) and 3′ (κE3′) enhancers was examined. Relative acetylation was determined by quantitative PCR and plotted as IP/input. The active G6PD and silent T4D loci serve as positive and negative controls, respectively.

Techniques Used: Real-time Polymerase Chain Reaction

53) Product Images from "Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes"

Article Title: Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes

Journal: Eurosurveillance

doi: 10.2807/1560-7917.ES.2018.23.6.17-00672

Multiplex PCR for detection of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 , European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) in the context of animal health and food safety, 2017
Figure Legend Snippet: Multiplex PCR for detection of mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 , European Union Reference Laboratory for Antimicrobial Resistance (EURL-AR) in the context of animal health and food safety, 2017

Techniques Used: Multiplex Assay, Polymerase Chain Reaction

54) Product Images from "Generation of Tumor-associated Cytotoxic T Lymphocytes Requires Interleukin 4 from CD8+ T Cells"

Article Title: Generation of Tumor-associated Cytotoxic T Lymphocytes Requires Interleukin 4 from CD8+ T Cells

Journal: The Journal of Experimental Medicine

doi:

The generation of CT26-specific CTLs does not require IL-4- or IL-4R-expression by CD4 +  T cells. SCID mice were reconstituted with sorted CD8 +  T cells from wt mice and sorted CD4 +  T cells from wt, IL-4 −/−  or IL-4R −/−  mice, respectively. Immunizations and in vitro restimulation of spleen cells were done as described in   Fig. 1 . CT26-specific (a and b) and AH1-specific CTL activity (c and d) of CD4 IL-4−/− /CD8 wt - (•) (a and c), CD4 IL-4R−/− /CD8 wt - (♦) (b and d), and CD4 wt /CD8 wt -recipients (▪) (a–d) was measured at various E:T ratios. Percent specific lysis represents the difference between percent lysis of CT26 cells and Renca cells (a and b) or the difference between percent lysis of AH1 peptide-loaded and control peptide loaded Renca cells (c and d). Splenocytes from nonimmunized CD4 wt /CD8 wt -recipients served as negative control (□).
Figure Legend Snippet: The generation of CT26-specific CTLs does not require IL-4- or IL-4R-expression by CD4 + T cells. SCID mice were reconstituted with sorted CD8 + T cells from wt mice and sorted CD4 + T cells from wt, IL-4 −/− or IL-4R −/− mice, respectively. Immunizations and in vitro restimulation of spleen cells were done as described in Fig. 1 . CT26-specific (a and b) and AH1-specific CTL activity (c and d) of CD4 IL-4−/− /CD8 wt - (•) (a and c), CD4 IL-4R−/− /CD8 wt - (♦) (b and d), and CD4 wt /CD8 wt -recipients (▪) (a–d) was measured at various E:T ratios. Percent specific lysis represents the difference between percent lysis of CT26 cells and Renca cells (a and b) or the difference between percent lysis of AH1 peptide-loaded and control peptide loaded Renca cells (c and d). Splenocytes from nonimmunized CD4 wt /CD8 wt -recipients served as negative control (□).

Techniques Used: Expressing, Mouse Assay, In Vitro, CTL Assay, Activity Assay, Lysis, Negative Control

IL-4- but not IL-4R-expression by CD8 +  T cells is required for the generation of CT26-specific CTLs. SCID mice were reconstituted with sorted CD4 +  T cells from wt mice and sorted CD8 +  T cells from wt, IL-4 −/− , or IL-4R −/−  mice, respectively. Recipients were immunized with CT26 and CT26-specific (a and b) as well as AH1-specific CTL activity (c and d) of CD4 wt /CD8 IL-4−/− - (•) (a and c), CD4 wt /CD8 IL-4R−/− - (♦) (b and d), and CD4 wt /CD8 wt -recipients (▪) (a–d) was determined as described in   Fig. 2 . Splenocytes from nonimmunized CD4 wt /CD8 wt -recipients served as negative control (□). The same controls are shown in a and   Fig. 2  a and in c and d because these experiments were done in parallel.
Figure Legend Snippet: IL-4- but not IL-4R-expression by CD8 + T cells is required for the generation of CT26-specific CTLs. SCID mice were reconstituted with sorted CD4 + T cells from wt mice and sorted CD8 + T cells from wt, IL-4 −/− , or IL-4R −/− mice, respectively. Recipients were immunized with CT26 and CT26-specific (a and b) as well as AH1-specific CTL activity (c and d) of CD4 wt /CD8 IL-4−/− - (•) (a and c), CD4 wt /CD8 IL-4R−/− - (♦) (b and d), and CD4 wt /CD8 wt -recipients (▪) (a–d) was determined as described in Fig. 2 . Splenocytes from nonimmunized CD4 wt /CD8 wt -recipients served as negative control (□). The same controls are shown in a and Fig. 2 a and in c and d because these experiments were done in parallel.

Techniques Used: Expressing, Mouse Assay, CTL Assay, Activity Assay, Negative Control

Naive CD8 +  T cells upregulate IL-4 gene expression within 24 h of antigen stimulation. Spleen cells from OT-I × RAG1 −/−  mice were incubated for 24 h in the presence (Δ) or absence (□) of 5 μg/ml peptide ova 257–264  (SIINFEKL) and the production of IL-4 mRNA was evaluated by quantitative RT-PCR. Amplification plots for IL-4 (a) and hypoxanthine guanine phosphoribosyltransferase (HPRT) (b) are shown for each RNA sample, in triplicates. IL-4 transcripts were normalized to HPRT abundance and analyzed in one reaction (multiplex analysis). One representative out of three experiments is shown. ΔRn represents changes of emission of reporter dye versus quenching dye over the course of the PCR reaction.
Figure Legend Snippet: Naive CD8 + T cells upregulate IL-4 gene expression within 24 h of antigen stimulation. Spleen cells from OT-I × RAG1 −/− mice were incubated for 24 h in the presence (Δ) or absence (□) of 5 μg/ml peptide ova 257–264 (SIINFEKL) and the production of IL-4 mRNA was evaluated by quantitative RT-PCR. Amplification plots for IL-4 (a) and hypoxanthine guanine phosphoribosyltransferase (HPRT) (b) are shown for each RNA sample, in triplicates. IL-4 transcripts were normalized to HPRT abundance and analyzed in one reaction (multiplex analysis). One representative out of three experiments is shown. ΔRn represents changes of emission of reporter dye versus quenching dye over the course of the PCR reaction.

Techniques Used: Expressing, Mouse Assay, Incubation, Quantitative RT-PCR, Amplification, Multiplex Assay, Polymerase Chain Reaction

CD4 +  T cell–dependent, CTL-mediated tumor immunity is impaired in IL-4 −/−  and IL-4R −/−  mice. (a) The indicated mice (5–6/group) were immunized with 10 6  irradiated CT26 cells, contralaterally challenged with 10 6  viable CT26 cells 2 wk later, and tumor growth was monitored. One out of three experiments with similar results is shown. (b) BALB/c mice (10/group) were immunized and challenged with CT26 as described in panel a and tumor growth was monitored. 2 d before challenge, mice were depleted of CD4 +  or CD8 +  T cells. For a and b, the percentage of tumor free mice 60 d after challenge is shown. (c) To determine tumor-specific CTL activity in vitro, BALB/c mice (five/group) were immunized twice with 10 6  irradiated CT26 cells in a 2-wk interval or left untreated. 7 to 10 d after the second immunization, spleen cells were restimulated for 7 d with the CT26-derived peptide AH1. CTL activity against CT26 was determined for immunized (diamonds) and nonimmunized BALB/c mice (triangles) at different E:T ratios. One representative experiment out of three is shown. (d) To test whether the generation of anti-CT26 CTLs was CD4 +  T cell dependent, SCID mice (four/group) were reconstituted with either sorted BALB/c CD4 +  and CD8 +  T cells or CD8 +  T cells only. Recipients were immunized or left untreated and analyzed for CTL activity as described in c. CTL activities of immunized CD4 wt /CD8 wt -recipients (squares), immunized CD8 wt -recipients (triangles), and nonimmunized CD4 wt /CD8 wt -recipients (circles) was measured against CT26.
Figure Legend Snippet: CD4 + T cell–dependent, CTL-mediated tumor immunity is impaired in IL-4 −/− and IL-4R −/− mice. (a) The indicated mice (5–6/group) were immunized with 10 6 irradiated CT26 cells, contralaterally challenged with 10 6 viable CT26 cells 2 wk later, and tumor growth was monitored. One out of three experiments with similar results is shown. (b) BALB/c mice (10/group) were immunized and challenged with CT26 as described in panel a and tumor growth was monitored. 2 d before challenge, mice were depleted of CD4 + or CD8 + T cells. For a and b, the percentage of tumor free mice 60 d after challenge is shown. (c) To determine tumor-specific CTL activity in vitro, BALB/c mice (five/group) were immunized twice with 10 6 irradiated CT26 cells in a 2-wk interval or left untreated. 7 to 10 d after the second immunization, spleen cells were restimulated for 7 d with the CT26-derived peptide AH1. CTL activity against CT26 was determined for immunized (diamonds) and nonimmunized BALB/c mice (triangles) at different E:T ratios. One representative experiment out of three is shown. (d) To test whether the generation of anti-CT26 CTLs was CD4 + T cell dependent, SCID mice (four/group) were reconstituted with either sorted BALB/c CD4 + and CD8 + T cells or CD8 + T cells only. Recipients were immunized or left untreated and analyzed for CTL activity as described in c. CTL activities of immunized CD4 wt /CD8 wt -recipients (squares), immunized CD8 wt -recipients (triangles), and nonimmunized CD4 wt /CD8 wt -recipients (circles) was measured against CT26.

Techniques Used: CTL Assay, Mouse Assay, Irradiation, Activity Assay, In Vitro, Derivative Assay

IL-4R-expression by non-T, non-B cells is required for the generation of CT26-specific CTLs. CD4 +  and CD8 +  T cells from wt mice were transferred to RAG2 −/− /IL-4R +/+  (▪) and RAG2 −/− /IL-4R −/−  mice (▴). The recipients were immunized with CT26 and CT26-specific (a) as well as AH1-specific CTL activity (b) was determined as described in   Fig. 2 . Splenocytes from reconstituted, nonimmunized RAG2 −/− /IL-4R +/+  recipients served as negative control (□).
Figure Legend Snippet: IL-4R-expression by non-T, non-B cells is required for the generation of CT26-specific CTLs. CD4 + and CD8 + T cells from wt mice were transferred to RAG2 −/− /IL-4R +/+ (▪) and RAG2 −/− /IL-4R −/− mice (▴). The recipients were immunized with CT26 and CT26-specific (a) as well as AH1-specific CTL activity (b) was determined as described in Fig. 2 . Splenocytes from reconstituted, nonimmunized RAG2 −/− /IL-4R +/+ recipients served as negative control (□).

Techniques Used: Expressing, Mouse Assay, CTL Assay, Activity Assay, Negative Control

55) Product Images from "EFFECTS OF COMBRETUM HEREROENSE AND CANTHIUMMUNDIANUM water EXTRACTS ON PRODUCTION AND EXPRESSION OF INTERLEUKIN-4"

Article Title: EFFECTS OF COMBRETUM HEREROENSE AND CANTHIUMMUNDIANUM water EXTRACTS ON PRODUCTION AND EXPRESSION OF INTERLEUKIN-4

Journal: African Journal of Traditional, Complementary, and Alternative Medicines

doi: 10.21010/ajtcam.v14i1.32

The activity index of Combretum hereroense (E1) and Canthium mundianum (E2) on the expression of IL-4 from two immunocompromised donor (F and F) at different concentrations of the plant extracts
Figure Legend Snippet: The activity index of Combretum hereroense (E1) and Canthium mundianum (E2) on the expression of IL-4 from two immunocompromised donor (F and F) at different concentrations of the plant extracts

Techniques Used: Activity Assay, Expressing

The activity index of Combretum hereroense (E1) and Canthium mundianum (E2) on the expression of IL-4 from three HIVnegative (A, B and C) individuals and one (D) HIVpositive donor at three different plant concentrations
Figure Legend Snippet: The activity index of Combretum hereroense (E1) and Canthium mundianum (E2) on the expression of IL-4 from three HIVnegative (A, B and C) individuals and one (D) HIVpositive donor at three different plant concentrations

Techniques Used: Activity Assay, Expressing

Examples of amplification curves (A) and melt curves (B) obtained from the amplification of the IL-4 cDNA from activated PBMCs exposed to the medicinal plant extracts from Combretum hereroense and Canthium mundianum .
Figure Legend Snippet: Examples of amplification curves (A) and melt curves (B) obtained from the amplification of the IL-4 cDNA from activated PBMCs exposed to the medicinal plant extracts from Combretum hereroense and Canthium mundianum .

Techniques Used: Amplification

The effects of Combretum hereroense (E1) and Canthium mundianum (E2) on the gene expression of IL-4 from immunocompromised individuals:
Figure Legend Snippet: The effects of Combretum hereroense (E1) and Canthium mundianum (E2) on the gene expression of IL-4 from immunocompromised individuals:

Techniques Used: Expressing

56) Product Images from "Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum"

Article Title: Structure of the type IV secretion system in different strains of Anaplasma phagocytophilum

Journal: BMC Genomics

doi: 10.1186/1471-2164-13-678

The 3 ′ end of A. phagocytophilum virB6 - 4 genes is composed of an unusually large tandem repeat region, which exhibits dramatic variability among strains. A . Map of the human HZ strain virB6 - 3 and virB6 - 4 genes, highlighting the location and structure of several repeat regions (R1-R4). The most variability occurred in R4; this region is 5.88 kb larger than previously reported for the Ap HZ genome (CP000235). The original sequence is diagrammed above the map, with the dashed line representing the segment missing in CP000235. Larger repeated R4 segments of 2.7 kb and 1.15 kb are indicated above. Vertical black bars within each gene designate segments encoding predicted transmembrane domains. BamHI sites, of which there is one in all R4 type 2 repeats (see Figure S2B), are indicated. Also shown are the positions of PCR primers used in C. B . BamHI genomic maps depicting the virB6 - 4 locus (black arrows).The segment encompassing R4 is highlighted below each respective map. In the regions outside the virB6 - 4 locus, corresponding BamHI fragments are shown in the same color. Overall, the optical map sizes were in good agreement with the actual sizes, except within R4. This is attributed to the limitation of optical mapping in resolving fragments
Figure Legend Snippet: The 3 ′ end of A. phagocytophilum virB6 - 4 genes is composed of an unusually large tandem repeat region, which exhibits dramatic variability among strains. A . Map of the human HZ strain virB6 - 3 and virB6 - 4 genes, highlighting the location and structure of several repeat regions (R1-R4). The most variability occurred in R4; this region is 5.88 kb larger than previously reported for the Ap HZ genome (CP000235). The original sequence is diagrammed above the map, with the dashed line representing the segment missing in CP000235. Larger repeated R4 segments of 2.7 kb and 1.15 kb are indicated above. Vertical black bars within each gene designate segments encoding predicted transmembrane domains. BamHI sites, of which there is one in all R4 type 2 repeats (see Figure S2B), are indicated. Also shown are the positions of PCR primers used in C. B . BamHI genomic maps depicting the virB6 - 4 locus (black arrows).The segment encompassing R4 is highlighted below each respective map. In the regions outside the virB6 - 4 locus, corresponding BamHI fragments are shown in the same color. Overall, the optical map sizes were in good agreement with the actual sizes, except within R4. This is attributed to the limitation of optical mapping in resolving fragments

Techniques Used: Sequencing, Polymerase Chain Reaction

57) Product Images from "Role of distinct surfaces of the G9a ankyrin repeat domain in histone and DNA methylation during embryonic stem cell self-renewal and differentiation"

Article Title: Role of distinct surfaces of the G9a ankyrin repeat domain in histone and DNA methylation during embryonic stem cell self-renewal and differentiation

Journal: Epigenetics & Chromatin

doi: 10.1186/1756-8935-7-27

G9a is not required for the transcriptional repression of selected early embryonic genes during retinoic acid (RA)-induced differentiation. The indicated mESC lines were untreated (0) or treated with 1 μM RA for 2, 4 or 8 days. (A) Immunoblots were used to determine G9a, Oct3/4 and GAPDH (loading control) protein levels in whole cell extracts. (B) Graph shows the messenger (mRNA) levels for the indicated genes using RT-qPCR analysis with Ubiquitin C (UBC) as the normalization control. All mRNA levels are normalized to the TT2 0 RA sample. Results shown are mean ± SD for three PCR reactions performed on the same cDNA sample and are representative of three independent experiments.
Figure Legend Snippet: G9a is not required for the transcriptional repression of selected early embryonic genes during retinoic acid (RA)-induced differentiation. The indicated mESC lines were untreated (0) or treated with 1 μM RA for 2, 4 or 8 days. (A) Immunoblots were used to determine G9a, Oct3/4 and GAPDH (loading control) protein levels in whole cell extracts. (B) Graph shows the messenger (mRNA) levels for the indicated genes using RT-qPCR analysis with Ubiquitin C (UBC) as the normalization control. All mRNA levels are normalized to the TT2 0 RA sample. Results shown are mean ± SD for three PCR reactions performed on the same cDNA sample and are representative of three independent experiments.

Techniques Used: Western Blot, Quantitative RT-PCR, Polymerase Chain Reaction

G9a, but not the histone H3 binding surface of the ankyrin repeat (ANK) domain, is required for efficient repression of SSEA-1 expression during embryonic stem cell (ESC) differentiation induced by retinoic acid (RA). Fluorescence-activated cell sorting (FACS) analysis of cell surface protein SSEA-1 expression in the indicated mESC lines that were untreated (0) or treated with RA for 3 or 7 days. Shaded histograms show nonspecific staining (obtained when staining exclusively with secondary antibody) and open histograms show specific SSEA-1 staining (staining with both primary and secondary antibodies).
Figure Legend Snippet: G9a, but not the histone H3 binding surface of the ankyrin repeat (ANK) domain, is required for efficient repression of SSEA-1 expression during embryonic stem cell (ESC) differentiation induced by retinoic acid (RA). Fluorescence-activated cell sorting (FACS) analysis of cell surface protein SSEA-1 expression in the indicated mESC lines that were untreated (0) or treated with RA for 3 or 7 days. Shaded histograms show nonspecific staining (obtained when staining exclusively with secondary antibody) and open histograms show specific SSEA-1 staining (staining with both primary and secondary antibodies).

Techniques Used: Binding Assay, Expressing, Fluorescence, FACS, Staining

Characterization of mouse embryonic stem (ES) clonal cell lines expressing G9a bearing point mutations in the cage and non-cage residues of the ankyrin repeat (ANK) domain. (A) Top panel: Diagram of full-length mG9a showing amino acid sequence numbers and specific domains: E , Glu-rich; Cys , Cys-rich ring finger-like; ANK , ankyrin repeat; SET , methyltransferase; and Pre and Post , Cys-rich Pre-SET and post-SET. Bottom panel: Schematic representation of the structural basis for ankyrin repeat (green) recognition of dimethylated histone H3 Lysine 9 (gray). G9a residue numbers are shown. H3K9me2 binds in a partial hydrophobic cage composed of residues highlighted in red. Peptide binding is further specified by the interaction of non-cage G9a residues (highlighted in purple) with H3 Ser10, Thr11, Gly12 and Gly13. (B) Immunoblots ( IB ) were performed on the indicated proteins (with GAPDH as loading control) using whole cell extracts derived from wild-type (TT2) mouse embryonic stem cells (mESCs), G9a -null mESCs (knock-out; KO) or distinct clonal ESC lines (indicated by #1 and #2) stably expressing wild-type G9a (+WT) or G9a harboring the indicated point mutations in the ANK domain.
Figure Legend Snippet: Characterization of mouse embryonic stem (ES) clonal cell lines expressing G9a bearing point mutations in the cage and non-cage residues of the ankyrin repeat (ANK) domain. (A) Top panel: Diagram of full-length mG9a showing amino acid sequence numbers and specific domains: E , Glu-rich; Cys , Cys-rich ring finger-like; ANK , ankyrin repeat; SET , methyltransferase; and Pre and Post , Cys-rich Pre-SET and post-SET. Bottom panel: Schematic representation of the structural basis for ankyrin repeat (green) recognition of dimethylated histone H3 Lysine 9 (gray). G9a residue numbers are shown. H3K9me2 binds in a partial hydrophobic cage composed of residues highlighted in red. Peptide binding is further specified by the interaction of non-cage G9a residues (highlighted in purple) with H3 Ser10, Thr11, Gly12 and Gly13. (B) Immunoblots ( IB ) were performed on the indicated proteins (with GAPDH as loading control) using whole cell extracts derived from wild-type (TT2) mouse embryonic stem cells (mESCs), G9a -null mESCs (knock-out; KO) or distinct clonal ESC lines (indicated by #1 and #2) stably expressing wild-type G9a (+WT) or G9a harboring the indicated point mutations in the ANK domain.

Techniques Used: Expressing, Sequencing, Binding Assay, Western Blot, Derivative Assay, Knock-Out, Stable Transfection

The G9a non-cage histone H3 binding surface of the ankyrin repeat (ANK) domain is required for de novo DNA methylation of the Oct3/4 gene promoter during retinoic acid (RA)-induced embryonic stem cell (ESC) differentiation. DNA extracted from the indicated mouse embryonic stem cell (mESC) lines, grown under self-renewal conditions or treated with RA for 8 days to induce differentiation, was treated with sodium bisulfite, amplified using specific Oct3/4 promoter primers, cloned and subjected to sequence analysis. (A) Schematic diagram of CpG positions within the Oct3/4 gene promoter is shown. The methylation status of eight CpG sites (no. 1 to 8) within the promoter (-90 to -258 relative to the transcription start site) was examined. (B) Percentage of DNA methylation for the Oct3/4 promoter region encompassing eight CpG sites of each clonal cell line is indicated in the table, which summarizes data shown in (C) and (D) . DNA extracted from the indicated mESC lines that were untreated (C) or treated with RA for 8 days (D) was treated with sodium bisulfite, amplified using specific Oct3/4 promoter primers, cloned and subjected to sequence analysis. Each horizontal row represents results from sequencing a separate DNA clone. The overall percentage of methylated CpG sites is indicated in parentheses beside the clone designation. The number of independent biological replicate experiments ( n ) from which the total number of DNA clones ( c ) were derived is also indicated.
Figure Legend Snippet: The G9a non-cage histone H3 binding surface of the ankyrin repeat (ANK) domain is required for de novo DNA methylation of the Oct3/4 gene promoter during retinoic acid (RA)-induced embryonic stem cell (ESC) differentiation. DNA extracted from the indicated mouse embryonic stem cell (mESC) lines, grown under self-renewal conditions or treated with RA for 8 days to induce differentiation, was treated with sodium bisulfite, amplified using specific Oct3/4 promoter primers, cloned and subjected to sequence analysis. (A) Schematic diagram of CpG positions within the Oct3/4 gene promoter is shown. The methylation status of eight CpG sites (no. 1 to 8) within the promoter (-90 to -258 relative to the transcription start site) was examined. (B) Percentage of DNA methylation for the Oct3/4 promoter region encompassing eight CpG sites of each clonal cell line is indicated in the table, which summarizes data shown in (C) and (D) . DNA extracted from the indicated mESC lines that were untreated (C) or treated with RA for 8 days (D) was treated with sodium bisulfite, amplified using specific Oct3/4 promoter primers, cloned and subjected to sequence analysis. Each horizontal row represents results from sequencing a separate DNA clone. The overall percentage of methylated CpG sites is indicated in parentheses beside the clone designation. The number of independent biological replicate experiments ( n ) from which the total number of DNA clones ( c ) were derived is also indicated.

Techniques Used: Binding Assay, DNA Methylation Assay, Amplification, Clone Assay, Sequencing, Methylation, Derivative Assay

Mutations in the histone H3 binding surface of the G9a ankyrin repeat (ANK) domain do not abrogate binding to DNMT3A. COS- 7 cells were transiently transfected with pcDNA3.1-mDNMT3A (A and B) or pSG5-HA-mGLP full-length (D) and pSG5-FLAG-mG9a full-length (A, B and D) or pSG5-HA-mG9a.ΔANK (B) . G9a was either wild-type (WT) or contained the indicated point mutations in the ANK domain. G9a, GLP and DNMT3A were immunoprecipitated from cell extracts with an anti-FLAG/anti-HA, anti-HA or anti-DNMT3A antibody, respectively. Non-immune IgG antibody was used for immunoprecipitation background estimation. Bound proteins were analyzed by immunoblot ( IB ) with the indicated antibodies. A 1% input sample was loaded for comparison. High (3 minutes) and low (1 minute) exposure times are shown (B) . (C) Immunoblots were used to determine H3K9me2 and H3 protein levels in acid histone extracts from the indicated undifferentiated mESC lines.
Figure Legend Snippet: Mutations in the histone H3 binding surface of the G9a ankyrin repeat (ANK) domain do not abrogate binding to DNMT3A. COS- 7 cells were transiently transfected with pcDNA3.1-mDNMT3A (A and B) or pSG5-HA-mGLP full-length (D) and pSG5-FLAG-mG9a full-length (A, B and D) or pSG5-HA-mG9a.ΔANK (B) . G9a was either wild-type (WT) or contained the indicated point mutations in the ANK domain. G9a, GLP and DNMT3A were immunoprecipitated from cell extracts with an anti-FLAG/anti-HA, anti-HA or anti-DNMT3A antibody, respectively. Non-immune IgG antibody was used for immunoprecipitation background estimation. Bound proteins were analyzed by immunoblot ( IB ) with the indicated antibodies. A 1% input sample was loaded for comparison. High (3 minutes) and low (1 minute) exposure times are shown (B) . (C) Immunoblots were used to determine H3K9me2 and H3 protein levels in acid histone extracts from the indicated undifferentiated mESC lines.

Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Western Blot

58) Product Images from "Mesenchymal stem cells and CXC chemokine receptor 4 overexpression improved the therapeutic effect on colitis via mucosa repair"

Article Title: Mesenchymal stem cells and CXC chemokine receptor 4 overexpression improved the therapeutic effect on colitis via mucosa repair

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.6233

CXCR-4 gene in BMSCs and CXCR-BMSCs. (A) Luciferase report gene absorbance in BMSCs and CXCR-BMSCs; (B-a) The melting curve of CXCR-4; (B-b) The relative expression of BMSCs and CXCR-BMSCs. *P
Figure Legend Snippet: CXCR-4 gene in BMSCs and CXCR-BMSCs. (A) Luciferase report gene absorbance in BMSCs and CXCR-BMSCs; (B-a) The melting curve of CXCR-4; (B-b) The relative expression of BMSCs and CXCR-BMSCs. *P

Techniques Used: Luciferase, Expressing

59) Product Images from "Molecular alterations in the TCR signaling pathway in patients with aplastic anemia"

Article Title: Molecular alterations in the TCR signaling pathway in patients with aplastic anemia

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-016-0261-6

The genotype characteristic of SNP rs231775 in CTLA-4 gene in AA and HIs. a Distribution of the genotype of SNP rs231775 in AA patients ( n = 67); b distribution of the genotype of SNP rs231775 in HIs ( n = 84); c agarose gel electrophoresis results for SNP rs231775 in CTLA-4 gene, lane 1 is the AG genotype, lane 2 is the GG genotype, lane 3 is a 50 bp DNA ladder, lane 4 is the AA genotype, and lane 5 is a PCR product without TseI digestion; d sequencing results of AA genotype; e sequencing results of AG genotype; f sequencing results of GG genotype; the red arrow indicates the single-nucleotide polymorphism sites
Figure Legend Snippet: The genotype characteristic of SNP rs231775 in CTLA-4 gene in AA and HIs. a Distribution of the genotype of SNP rs231775 in AA patients ( n = 67); b distribution of the genotype of SNP rs231775 in HIs ( n = 84); c agarose gel electrophoresis results for SNP rs231775 in CTLA-4 gene, lane 1 is the AG genotype, lane 2 is the GG genotype, lane 3 is a 50 bp DNA ladder, lane 4 is the AA genotype, and lane 5 is a PCR product without TseI digestion; d sequencing results of AA genotype; e sequencing results of AG genotype; f sequencing results of GG genotype; the red arrow indicates the single-nucleotide polymorphism sites

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing

Correlation analysis of the CTLA-4 and Cbl-b expression levels for different genotypes of SNP rs231775 in CTLA-4 gene in AA and HIs. a CTLA-4 and Cbl-b in AA with the AA genotype, b CTLA-4 and Cbl-b in AA with the AG genotype, c CTLA-4 and Cbl-b in AA with the GG genotype, d CTLA-4 and Cbl-b in HIs with the AA genotype, e CTLA-4 and Cbl-b in HIs with the AG genotype, and f CTLA-4 and Cbl-b in HIs with the GG genotype
Figure Legend Snippet: Correlation analysis of the CTLA-4 and Cbl-b expression levels for different genotypes of SNP rs231775 in CTLA-4 gene in AA and HIs. a CTLA-4 and Cbl-b in AA with the AA genotype, b CTLA-4 and Cbl-b in AA with the AG genotype, c CTLA-4 and Cbl-b in AA with the GG genotype, d CTLA-4 and Cbl-b in HIs with the AA genotype, e CTLA-4 and Cbl-b in HIs with the AG genotype, and f CTLA-4 and Cbl-b in HIs with the GG genotype

Techniques Used: Expressing

60) Product Images from "The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription"

Article Title: The mutated tegument protein UL7 attenuates the virulence of herpes simplex virus 1 by reducing the modulation of α-4 gene transcription

Journal: Virology Journal

doi: 10.1186/s12985-016-0600-9

The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P
Figure Legend Snippet: The UL7-MU viral strain exhibits attenuated phenotypes in a latent mouse infection model compared with the WT strain. a BALB/c mice were infected with WT HSV-1, UL7-MU or PBS via the foot pad at a dose of 5x10 3 /10 μl per mouse. The viral load was detected in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles) by absolute real-time RT-PCR. Viral copy numbers were quantified according to the HSV-1 DNA standard pGM-T UL30 plasmid. b The levels of LAT expression in the CNS of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles), as determined by relative real-time RT-PCR. The graphic indicates the fold change of RNA levels in virus-infected mice compared to PBS-injected mice. The mouse housekeeping gene GAPDH was used to normalize quantities in mouse tissue. Relative quantification was performed by the comparative Ct method (△△Ct) using RNA from PBS mice as a calibrator. c Viral load detection in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). d The levels of LAT expression in the spinal cord of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). e Viral load detection in the trigeminal nerve of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). f The levels of LAT expression in the trigeminal nerves of mice challenged with WT (open boxes), UL7-MU (filled circles) or PBS (filled triangles). Data are shown as the means ± SD (experiments done once in triplicate). ∗∗∗ P

Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR, Plasmid Preparation, Expressing, Injection

UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P
Figure Legend Snippet: UL7 protein is involved in regulating the transcriptional activation of the HSV-1 α-4 gene. a Comparison of α-4 transcriptional efficiencies during the proliferetion of UL7-MU (filled circles) and WT viral strains (open boxes). Gene expression levels were detected using absolute real-time RT-PCR. Gene copy numbers were quantified according to the gene RNA standard. b HEK293T cells were co-transfected with pGL-α-4, pGL-UL23 or pGL-UL41 and UL7-WT, UL7-MU or control plasmid for 36 h before luciferase activities were quantified. Data are shown as means ± SD. ∗∗ P

Techniques Used: Activation Assay, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase

Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box
Figure Legend Snippet: Identification of the mutated UL7 gene in the viral strain. a Design of the g-RNA sequences for UL7 gene mutation, with the target sites of the g-RNAs (UL7-1 and UL7-2) labeled in yellow. The fragments amplified by oligo 1 and oligo 2 were used in the SURVEYOR assay. b SURVEYOR detection of the mutated genes. Top: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 (P1); bottom: SURVEYOR assay of HSV-1 genomic DNA extracted from HEK293T cells expressing UL7-1 and UL7-2 individually or together infected with HSV-1 progeny virus (P2). c Identification of the mutated UL7 gene in the viral strain. The UL7 mutant was identified via PCR using UL7-sense and UL7-antisense primers. The mutated UL7 gene is indicated with a red box

Techniques Used: Mutagenesis, Labeling, Amplification, Expressing, Infection, Polymerase Chain Reaction

61) Product Images from "Generation of Induced Pluripotent Stem Cells in Rabbits"

Article Title: Generation of Induced Pluripotent Stem Cells in Rabbits

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.150540

Characterization of rabbit iPS cells by their gene expression, telomerase activity, and DNA methylation of the OCT3/4 promoter. A , GFP fluorescence signal observed in the cells at 7 days after infection (iPS-L day 7 and iPS-S day 7). However, it became undetectable in iPS-L1 cells and iPS-S1 cells as early as passages 3 and 2, respectively. B , RT-PCR analysis of the expression of selected pluripotency-related genes and transgenes ( Tgs ) in rabbit ES cells, rabbit iPS cells, the original rabbit liver and stomach cells, and liver cells at 7 days after infection ( iPS-L day 7 ). C , detection of the telomerase activity of rabbit iPS cells using the telomeric repeat amplification protocol. The iPS cells showed high telomerase activity, similar to that of rabbit ES cells. Heat-inactivated samples (+) were used as negative controls. N.C ., negative control with lysis buffer only. D , bisulfite genomic sequencing of CpG-enriched regions of the rabbit OCT3/4 promoter in iPS cells and their original (parental) somatic cells. Both liver-derived ( iPS-L1 and 2 , passage 7) and stomach cell-derived ( iPS-S1 and 2 , passage 6) lines showed a highly unmethylated pattern whereas their parental cells showed a highly methylated pattern. Open circles and filled circles represent unmethylated and methylated CpG sites, respectively. Scale bar , 100 μm.
Figure Legend Snippet: Characterization of rabbit iPS cells by their gene expression, telomerase activity, and DNA methylation of the OCT3/4 promoter. A , GFP fluorescence signal observed in the cells at 7 days after infection (iPS-L day 7 and iPS-S day 7). However, it became undetectable in iPS-L1 cells and iPS-S1 cells as early as passages 3 and 2, respectively. B , RT-PCR analysis of the expression of selected pluripotency-related genes and transgenes ( Tgs ) in rabbit ES cells, rabbit iPS cells, the original rabbit liver and stomach cells, and liver cells at 7 days after infection ( iPS-L day 7 ). C , detection of the telomerase activity of rabbit iPS cells using the telomeric repeat amplification protocol. The iPS cells showed high telomerase activity, similar to that of rabbit ES cells. Heat-inactivated samples (+) were used as negative controls. N.C ., negative control with lysis buffer only. D , bisulfite genomic sequencing of CpG-enriched regions of the rabbit OCT3/4 promoter in iPS cells and their original (parental) somatic cells. Both liver-derived ( iPS-L1 and 2 , passage 7) and stomach cell-derived ( iPS-S1 and 2 , passage 6) lines showed a highly unmethylated pattern whereas their parental cells showed a highly methylated pattern. Open circles and filled circles represent unmethylated and methylated CpG sites, respectively. Scale bar , 100 μm.

Techniques Used: Expressing, Activity Assay, DNA Methylation Assay, Fluorescence, Infection, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Lysis, Genomic Sequencing, Derivative Assay, Methylation

Expression of pluripotency markers in rabbit iPS cells detected by staining for alkaline phosphatase ( AP ) activity and by immunostaining. The iPS cells were positive for alkaline phosphatase, SSEA1, SSEA4, OCT3/4, and NANOG, but not for SSEA3. Representative images were prepared from iPS-L1 cells at passage 12. Scale bars , 100 μm.
Figure Legend Snippet: Expression of pluripotency markers in rabbit iPS cells detected by staining for alkaline phosphatase ( AP ) activity and by immunostaining. The iPS cells were positive for alkaline phosphatase, SSEA1, SSEA4, OCT3/4, and NANOG, but not for SSEA3. Representative images were prepared from iPS-L1 cells at passage 12. Scale bars , 100 μm.

Techniques Used: Expressing, Staining, Activity Assay, Immunostaining

62) Product Images from "Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana"

Article Title: Improving agroinfiltration-based transient gene expression in Nicotiana benthamiana

Journal: Plant Methods

doi: 10.1186/s13007-018-0343-2

Effects of co‐expressing virus‐derived genes encoding cell cycle regulatory proteins. Agrobacteria strain AGL1 harbouring pEAQ‐GSN were co‐infiltrated into N. benthamiana leaves with a TYDV Rep / RepA or RepA genes and the TYDV RepA gene containing a LxCxK mutation in the RB-binding motif, and b cell cycle regulatory genes from related circular ssDNA plant viruses, under the transcriptional control of the truncated CaMV 35S (− 90) promoter (Δ35S). Leaves were sampled 4 dpi and TSP extracted for GUS fluorometric enzyme assays. Columns represent relative levels of mean GUS enzyme activities and bars represent ± SE. (†) indicates the reference treatment and (*) indicates data significantly different to the reference ( p
Figure Legend Snippet: Effects of co‐expressing virus‐derived genes encoding cell cycle regulatory proteins. Agrobacteria strain AGL1 harbouring pEAQ‐GSN were co‐infiltrated into N. benthamiana leaves with a TYDV Rep / RepA or RepA genes and the TYDV RepA gene containing a LxCxK mutation in the RB-binding motif, and b cell cycle regulatory genes from related circular ssDNA plant viruses, under the transcriptional control of the truncated CaMV 35S (− 90) promoter (Δ35S). Leaves were sampled 4 dpi and TSP extracted for GUS fluorometric enzyme assays. Columns represent relative levels of mean GUS enzyme activities and bars represent ± SE. (†) indicates the reference treatment and (*) indicates data significantly different to the reference ( p

Techniques Used: Expressing, Derivative Assay, Mutagenesis, Binding Assay

Schematic representation of pEAQ-GSN, pSPECIAL and pNEEDS vectors. pEAQ-GSN was assembled by introducing the uid ]. pSPECIAL is based on pEAQ-GSN with a downstream expression cassette encoding the truncated CMV 2b (1-94) silencing suppressor protein. pNEEDS is a pBIN-Plus binary vector comprising two expression cassettes encoding the AtBAG 4 stress tolerance protein under the transcriptional control of the nos promoter and the TYDV Rep/RepA cell cycle control gene products under the transcriptional control of the truncated CaMV (− 90) promoter. 35SP = Cauliflower mosaic virus 35S promoter; CPMV 5′ UTR = Cowpea mosaic virus RNA‐2 5′UTR; uid A = gene encoding GUS; syntron = synthetic intron; CPMV 3′ UTR = Cowpea mosaic virus RNA‐2 3′UTR; nosT = nopaline synthase terminator from Agrobacterium ; TBSV p19 = Tomato bushy stunt virus p19 silencing suppressor gene; 35ST = Cauliflower mosaic virus 35S terminator; CMV 2b (1-94) = Cucumber mosaic virus truncated 2b silencing suppressor gene (amino acids 1-94); nosP = nopaline synthase promoter from Agrobacterium ; AtBAG 4= Arabidopsis BAG 4 gene; ∆35SP = truncated Cauliflower mosaic virus 35S (− 90) promoter; TYDV Rep / RepA = Tobacco yellow dwarf virus Rep / RepA gene encoding both Rep and RepA
Figure Legend Snippet: Schematic representation of pEAQ-GSN, pSPECIAL and pNEEDS vectors. pEAQ-GSN was assembled by introducing the uid ]. pSPECIAL is based on pEAQ-GSN with a downstream expression cassette encoding the truncated CMV 2b (1-94) silencing suppressor protein. pNEEDS is a pBIN-Plus binary vector comprising two expression cassettes encoding the AtBAG 4 stress tolerance protein under the transcriptional control of the nos promoter and the TYDV Rep/RepA cell cycle control gene products under the transcriptional control of the truncated CaMV (− 90) promoter. 35SP = Cauliflower mosaic virus 35S promoter; CPMV 5′ UTR = Cowpea mosaic virus RNA‐2 5′UTR; uid A = gene encoding GUS; syntron = synthetic intron; CPMV 3′ UTR = Cowpea mosaic virus RNA‐2 3′UTR; nosT = nopaline synthase terminator from Agrobacterium ; TBSV p19 = Tomato bushy stunt virus p19 silencing suppressor gene; 35ST = Cauliflower mosaic virus 35S terminator; CMV 2b (1-94) = Cucumber mosaic virus truncated 2b silencing suppressor gene (amino acids 1-94); nosP = nopaline synthase promoter from Agrobacterium ; AtBAG 4= Arabidopsis BAG 4 gene; ∆35SP = truncated Cauliflower mosaic virus 35S (− 90) promoter; TYDV Rep / RepA = Tobacco yellow dwarf virus Rep / RepA gene encoding both Rep and RepA

Techniques Used: Expressing, Plasmid Preparation

63) Product Images from "Correlation between IL-4 and IL-13 gene polymorphisms and asthma in Uygur children in Xinjiang"

Article Title: Correlation between IL-4 and IL-13 gene polymorphisms and asthma in Uygur children in Xinjiang

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.7096

Electrophoretogram of IL-13 Arg130Gln PCR product. M: marker; 1–21 indicate part of the objects of study.
Figure Legend Snippet: Electrophoretogram of IL-13 Arg130Gln PCR product. M: marker; 1–21 indicate part of the objects of study.

Techniques Used: Polymerase Chain Reaction, Marker

64) Product Images from "Staphylococcus aureus enterotoxins modulate IL-22-secreting cells in adults with atopic dermatitis"

Article Title: Staphylococcus aureus enterotoxins modulate IL-22-secreting cells in adults with atopic dermatitis

Journal: Scientific Reports

doi: 10.1038/s41598-018-25125-0

Cytokine gene expression profiles in AD skin. Expression profiles of IL-22, IL-4, IL-10, and TNF-α in skin of the healthy control group (HC, n = 5) compared with AD patients (AD, n = 10), assessed by real-time PCR. Lines represent medians with interquartile ranges of cytokines in skin specimens. *p ≤ 0.05 and **p ≤ 0.01.
Figure Legend Snippet: Cytokine gene expression profiles in AD skin. Expression profiles of IL-22, IL-4, IL-10, and TNF-α in skin of the healthy control group (HC, n = 5) compared with AD patients (AD, n = 10), assessed by real-time PCR. Lines represent medians with interquartile ranges of cytokines in skin specimens. *p ≤ 0.05 and **p ≤ 0.01.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

65) Product Images from "Identification of a Secreted Casein Kinase 1 in Leishmania donovani: Effect of Protein over Expression on Parasite Growth and Virulence"

Article Title: Identification of a Secreted Casein Kinase 1 in Leishmania donovani: Effect of Protein over Expression on Parasite Growth and Virulence

Journal: PLoS ONE

doi: 10.1371/journal.pone.0079287

Comparison of wild-type and mutant promastigote growth and metacyclogenesis in culture. Promastigotes, Ld:wild type (wt), Ld:LUC (luc) and Ld:CK1.4-FLAG (CK1.4) expressing mutants, were diluted (10 5 cells/ml) at day 0 in complete culture media. Panel A. Parasite growth was monitored daily by counting live cells in a haemocytometer. All experiments were performed in triplicates. *, P
Figure Legend Snippet: Comparison of wild-type and mutant promastigote growth and metacyclogenesis in culture. Promastigotes, Ld:wild type (wt), Ld:LUC (luc) and Ld:CK1.4-FLAG (CK1.4) expressing mutants, were diluted (10 5 cells/ml) at day 0 in complete culture media. Panel A. Parasite growth was monitored daily by counting live cells in a haemocytometer. All experiments were performed in triplicates. *, P

Techniques Used: Mutagenesis, Expressing

Expression of CK1.4-FLAG by mutant L. donovani promastigotes (Ld:CK1.4-FLAG). SDS-PAGE and Western blot analysis of total cellular lysates (10 7 promastigotes/lane) from wild type (wt) and Ld:CK1.4-FLAG (CK1.4) promastigotes using Panel A: anti-FLAG M2 monoclonal antibody (1/1000 dilution, Sigma - Aldrich Chemical Co.) or Panel B: rabbit polyclonal anti-HSP83 (1/5000 dilution) or anti-CK1.4 (1/7500 dilution).
Figure Legend Snippet: Expression of CK1.4-FLAG by mutant L. donovani promastigotes (Ld:CK1.4-FLAG). SDS-PAGE and Western blot analysis of total cellular lysates (10 7 promastigotes/lane) from wild type (wt) and Ld:CK1.4-FLAG (CK1.4) promastigotes using Panel A: anti-FLAG M2 monoclonal antibody (1/1000 dilution, Sigma - Aldrich Chemical Co.) or Panel B: rabbit polyclonal anti-HSP83 (1/5000 dilution) or anti-CK1.4 (1/7500 dilution).

Techniques Used: Expressing, Mutagenesis, SDS Page, Western Blot

Multi-alignment of leishmanial CK1.4 protein sequences. Predicted protein sequence for CK1.4 orthologs from Leishmania major (Lmaj - Gene Bank Accession No. CBZ12245.1), L. donovani (Ldon - JN225463), L. infantum (Linf - CAM69136.1), L. mexicana (Lmex - CBZ28278.1) and L. braziliensis (Lbra - CAM45467.1). Identical amino acids are marked by dots, and the gaps by red dashes. The blue brackets delineate the conserved protein kinase domain.
Figure Legend Snippet: Multi-alignment of leishmanial CK1.4 protein sequences. Predicted protein sequence for CK1.4 orthologs from Leishmania major (Lmaj - Gene Bank Accession No. CBZ12245.1), L. donovani (Ldon - JN225463), L. infantum (Linf - CAM69136.1), L. mexicana (Lmex - CBZ28278.1) and L. braziliensis (Lbra - CAM45467.1). Identical amino acids are marked by dots, and the gaps by red dashes. The blue brackets delineate the conserved protein kinase domain.

Techniques Used: Sequencing

Time course of CK1.4 release by Leishmania donovani promastigotes. Cell-free supernatants were prepared from Ld:wt and/or Ld:CK1.4-FLAG parasites at different times (min) post-induction with ionomycin (5 µM)/EGTA (1 mM), and examined by SDS-PAGE - Western blotting. Panel A: Membranes were incubated with anti-FLAG M2 monoclonal antibody (1/1000 dilution, Sigma –Aldrich Chemical Co); Lysate is a positive control prepared from 10 7 Ld:CK1.4-FLAG promastigotes collected at t = 0. Panel B: Membranes probed simultaneously with rabbit polyclonal anti-CK1.4 (1/7500 dilution) and anti-KMP 11 (1/2000 dilution) antibodies. Lanes labeled Lysate were prepared from either 10 7 Ld:wt or Ld:CK1.4 promastigotes collected at t = 0 min. Antibody binding was detected by incubation of the membrane with a chemiluminescent substrate and exposure to X-ray film.
Figure Legend Snippet: Time course of CK1.4 release by Leishmania donovani promastigotes. Cell-free supernatants were prepared from Ld:wt and/or Ld:CK1.4-FLAG parasites at different times (min) post-induction with ionomycin (5 µM)/EGTA (1 mM), and examined by SDS-PAGE - Western blotting. Panel A: Membranes were incubated with anti-FLAG M2 monoclonal antibody (1/1000 dilution, Sigma –Aldrich Chemical Co); Lysate is a positive control prepared from 10 7 Ld:CK1.4-FLAG promastigotes collected at t = 0. Panel B: Membranes probed simultaneously with rabbit polyclonal anti-CK1.4 (1/7500 dilution) and anti-KMP 11 (1/2000 dilution) antibodies. Lanes labeled Lysate were prepared from either 10 7 Ld:wt or Ld:CK1.4 promastigotes collected at t = 0 min. Antibody binding was detected by incubation of the membrane with a chemiluminescent substrate and exposure to X-ray film.

Techniques Used: SDS Page, Western Blot, Incubation, Positive Control, Labeling, Binding Assay

Localization of CK1.4 in Leishmania donovani promastigotes by immunefluorescent staining with anti-FLAG and anti-CK1.4 antibodies. Logarithmic stage Ld:CK1.4-FLAG (Panels Ld:CK1.4) and Ld:LUC (Panels Ld:luc) promastigotes were fixed in 4% paraformaldehyde, centrifuged onto poly-L-lysine coated slides, and permeabilized with ice cold methanol. The slides were incubated with anti-FLAG M2 monoclonal antibodies (1/500 dilution), rabbit anti-CK1.4 polyclonal antibodies (1/7500 dilution), or buffer alone. Staining was carried out by incubating slides in appropriate secondary antibody, either Cy2-goat anti-mouse IgG or Cy3-goat anti-rabbit IgG (Panel Cy2/3; 1/200 dilution), and mounted in Fluoroshield with DAPI (Panel DAPI). The immunofluorescence examined using an Apochromat oil immersion objective (100×magnification) on an Olympus IX71S8F microscope.
Figure Legend Snippet: Localization of CK1.4 in Leishmania donovani promastigotes by immunefluorescent staining with anti-FLAG and anti-CK1.4 antibodies. Logarithmic stage Ld:CK1.4-FLAG (Panels Ld:CK1.4) and Ld:LUC (Panels Ld:luc) promastigotes were fixed in 4% paraformaldehyde, centrifuged onto poly-L-lysine coated slides, and permeabilized with ice cold methanol. The slides were incubated with anti-FLAG M2 monoclonal antibodies (1/500 dilution), rabbit anti-CK1.4 polyclonal antibodies (1/7500 dilution), or buffer alone. Staining was carried out by incubating slides in appropriate secondary antibody, either Cy2-goat anti-mouse IgG or Cy3-goat anti-rabbit IgG (Panel Cy2/3; 1/200 dilution), and mounted in Fluoroshield with DAPI (Panel DAPI). The immunofluorescence examined using an Apochromat oil immersion objective (100×magnification) on an Olympus IX71S8F microscope.

Techniques Used: Staining, Incubation, Immunofluorescence, Microscopy

Expression of His-tag CK1.4 wild-type and deletion constructs. Panel A. Schematic description of recombinant full-length CK1.4 and truncated polypeptides. Conserved protein kinase catalytic domain – speckled box; predicted signal polypeptide – solid black box; (His) 6 epitope tag – his ; Oligonucleotide primers used for PCR: forward primers -MFCK1 and PFCK1; reverse primers – MRCK1 and P2MRCK1. Predicted molecular mass (kDa) of each recombinant protein (A – D) and casein kinase activity (rCK1) is given. Panel B. Western blot analysis of full length CK1.4 and truncated polypeptides expressed in E. coli. Expression of recombinant proteins was induced by addition of L-arabinose and IPTG for 18 hrs at 22°C (Lanes +). Non-induced bacteria were used as negative controls (Lanes −). Bacterial lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with Nickel conjugated Horseradish peroxidase (1/10000 dilution, HisDetector Nickel-HRP, KPL Inc, USA). Binding was detected using chemiluminescent substrate. His tagged recombinant proteins in Lanes: A - LdCK1.4; B - LdCK1.4Δ 1–90 ; C - LdCK1.4Δ 411–566 , and D - LdCK1.4Δ 1–90,411–566 .
Figure Legend Snippet: Expression of His-tag CK1.4 wild-type and deletion constructs. Panel A. Schematic description of recombinant full-length CK1.4 and truncated polypeptides. Conserved protein kinase catalytic domain – speckled box; predicted signal polypeptide – solid black box; (His) 6 epitope tag – his ; Oligonucleotide primers used for PCR: forward primers -MFCK1 and PFCK1; reverse primers – MRCK1 and P2MRCK1. Predicted molecular mass (kDa) of each recombinant protein (A – D) and casein kinase activity (rCK1) is given. Panel B. Western blot analysis of full length CK1.4 and truncated polypeptides expressed in E. coli. Expression of recombinant proteins was induced by addition of L-arabinose and IPTG for 18 hrs at 22°C (Lanes +). Non-induced bacteria were used as negative controls (Lanes −). Bacterial lysates were separated by SDS-PAGE, transferred to nitrocellulose membranes and probed with Nickel conjugated Horseradish peroxidase (1/10000 dilution, HisDetector Nickel-HRP, KPL Inc, USA). Binding was detected using chemiluminescent substrate. His tagged recombinant proteins in Lanes: A - LdCK1.4; B - LdCK1.4Δ 1–90 ; C - LdCK1.4Δ 411–566 , and D - LdCK1.4Δ 1–90,411–566 .

Techniques Used: Expressing, Construct, Recombinant, Polymerase Chain Reaction, Activity Assay, Western Blot, SDS Page, Binding Assay

66) Product Images from "Enhanced Expression of Tissue Inhibitor of Metalloproteinases-4 Gene in Human Osteoarthritic Synovial Membranes and Its Differential Regulation by Cytokines in Chondrocytes"

Article Title: Enhanced Expression of Tissue Inhibitor of Metalloproteinases-4 Gene in Human Osteoarthritic Synovial Membranes and Its Differential Regulation by Cytokines in Chondrocytes

Journal: The Open Rheumatology Journal

doi: 10.2174/1874312901105010081

A ) Expression of the TIMP-4 gene in human synovial membranes from non-arthritic and osteoarthritic patients. The specific amplification of TIMP-4 and GAPDH fragments (arrows) along with the lambda Hind III or 100 bp ladder markers is shown. ‘No DNA’ control lane represents PCR reactions with primers but without cDNA.  B ) Middle panels show two graphic representations of semi-quantitative analysis of TIMP-4 RNA bands. *** p
Figure Legend Snippet: A ) Expression of the TIMP-4 gene in human synovial membranes from non-arthritic and osteoarthritic patients. The specific amplification of TIMP-4 and GAPDH fragments (arrows) along with the lambda Hind III or 100 bp ladder markers is shown. ‘No DNA’ control lane represents PCR reactions with primers but without cDNA. B ) Middle panels show two graphic representations of semi-quantitative analysis of TIMP-4 RNA bands. *** p

Techniques Used: Expressing, Amplification, Polymerase Chain Reaction

67) Product Images from "CLASS2: accurate and efficient splice variant annotation from RNA-seq reads"

Article Title: CLASS2: accurate and efficient splice variant annotation from RNA-seq reads

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw158

PCR validation of CLASS2 output. ( A ) PCR validation strategy: blue squares represent annotated exons, the red rectangle represents the identified intron retention event, and the blue lines with arrowheads represent introns. Green arrows denote the location of the PCR primers. Human blood cDNA and genomic DNA were amplified with primer sets targeting intron retention events in ( B ) CACNA2D4 and ( D ) KLRF1 genes. For each primer set, a strong PCR product of the expected size was observed in cDNA but not genomic DNA. The sequences of the PCR reactions for ( C ) CACNA2D4 and ( E ) KLRF1 , labeled ‘YourSeq’ in the figure, were aligned against the human genome using the UCSC Genome Browser.
Figure Legend Snippet: PCR validation of CLASS2 output. ( A ) PCR validation strategy: blue squares represent annotated exons, the red rectangle represents the identified intron retention event, and the blue lines with arrowheads represent introns. Green arrows denote the location of the PCR primers. Human blood cDNA and genomic DNA were amplified with primer sets targeting intron retention events in ( B ) CACNA2D4 and ( D ) KLRF1 genes. For each primer set, a strong PCR product of the expected size was observed in cDNA but not genomic DNA. The sequences of the PCR reactions for ( C ) CACNA2D4 and ( E ) KLRF1 , labeled ‘YourSeq’ in the figure, were aligned against the human genome using the UCSC Genome Browser.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling

68) Product Images from "Gene and protein expression of a soluble form of CTLA-4 in a healthy dog"

Article Title: Gene and protein expression of a soluble form of CTLA-4 in a healthy dog

Journal: The Journal of Veterinary Medical Science

doi: 10.1292/jvms.16-0583

RT-PCR amplification of canine CTLA-4 mRNA. Bands corresponding to 450 and 550 bp were observed. M; 100 bp DNA maker.
Figure Legend Snippet: RT-PCR amplification of canine CTLA-4 mRNA. Bands corresponding to 450 and 550 bp were observed. M; 100 bp DNA maker.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification

69) Product Images from "The Effects of Antidepressants “Fluoxetine and Imipramine” on Vascular Abnormalities and Toll Like Receptor-4 Expression in Diabetic and Non-Diabetic Rats Exposed to Chronic Stress"

Article Title: The Effects of Antidepressants “Fluoxetine and Imipramine” on Vascular Abnormalities and Toll Like Receptor-4 Expression in Diabetic and Non-Diabetic Rats Exposed to Chronic Stress

Journal: PLoS ONE

doi: 10.1371/journal.pone.0120559

Effects of fluoxetine (FLU) versus imipramine (IMIP) on body weight in non-diabetic (A) and diabetic (B) rats exposed to chronic restraint stress (CRS). Data are mean±SEM of 9–14 animals per group **P
Figure Legend Snippet: Effects of fluoxetine (FLU) versus imipramine (IMIP) on body weight in non-diabetic (A) and diabetic (B) rats exposed to chronic restraint stress (CRS). Data are mean±SEM of 9–14 animals per group **P

Techniques Used:

Effects of fluoxetine (FLU) versus imipramine (IMIP) on systolic blood pressure (SBP) in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM (n = 6). ***P
Figure Legend Snippet: Effects of fluoxetine (FLU) versus imipramine (IMIP) on systolic blood pressure (SBP) in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM (n = 6). ***P

Techniques Used:

Effects of fluoxetine (FLU) versus imipramine (IMIP) on % phenylephrine (PhE)-induced contraction in non-diabetic (A) diabetic rats (B) and maximal relaxation response (%) of acetylcholine in non-diabetic (C) diabetic rats (D) exposed to chronic restraint stress (CRS). Data are mean±SEM; (n = 6). *P
Figure Legend Snippet: Effects of fluoxetine (FLU) versus imipramine (IMIP) on % phenylephrine (PhE)-induced contraction in non-diabetic (A) diabetic rats (B) and maximal relaxation response (%) of acetylcholine in non-diabetic (C) diabetic rats (D) exposed to chronic restraint stress (CRS). Data are mean±SEM; (n = 6). *P

Techniques Used:

TNF-α immunohistochemical staining (IHCX400) of aortic sections in different experimental groups. (A) Control non-diabetic group with focal faint TNF-a immunostaining (brownish color), (B) Non-diabetic/CRS vehicle-treated group shows moderate diffuse immunostaining, (C) Non-diabetic/CRS fluoxetine-treated and (D) Non-diabetic/CRS imipramine-treated groups show focal mild staining, (E) Control diabetic group (F) Diabetic/CRS vehicle-treated gr oup shows strong diffuse immunostaining, (G) Diabetic/CRS fluoxetine-treated group shows focal mild staining and (H) Diabetic/CRS imipramine-treated group show moderate diffuse immunostaining.
Figure Legend Snippet: TNF-α immunohistochemical staining (IHCX400) of aortic sections in different experimental groups. (A) Control non-diabetic group with focal faint TNF-a immunostaining (brownish color), (B) Non-diabetic/CRS vehicle-treated group shows moderate diffuse immunostaining, (C) Non-diabetic/CRS fluoxetine-treated and (D) Non-diabetic/CRS imipramine-treated groups show focal mild staining, (E) Control diabetic group (F) Diabetic/CRS vehicle-treated gr oup shows strong diffuse immunostaining, (G) Diabetic/CRS fluoxetine-treated group shows focal mild staining and (H) Diabetic/CRS imipramine-treated group show moderate diffuse immunostaining.

Techniques Used: Immunohistochemistry, Staining, Immunostaining

Effects of fluoxetine (FLU) and imipramine (IMIP) on percentage of initial blood glucose level during insulin tolerance test in non-diabetic (A) and diabetic (B) rats exposed to chronic restraint stress (CRS). Data are mean±SEM; (n = 6).*P
Figure Legend Snippet: Effects of fluoxetine (FLU) and imipramine (IMIP) on percentage of initial blood glucose level during insulin tolerance test in non-diabetic (A) and diabetic (B) rats exposed to chronic restraint stress (CRS). Data are mean±SEM; (n = 6).*P

Techniques Used:

(A) Ethidium bromide-stained agarose gel electrophoresis showing the amplified RT-PCR products of TLR-4 (299bp) and β-actin (180 bp) as an internal standard, from aortic homogenates of Wistar rats. First Lane (MW): molecular weight ladder standard. Lane 1: control non-diabetic group. Lane 2: Non-diabetic/CRS vehicle-treated group. Lane 3: Non-diabetic/CRS fluoxetine-treated group. Lane 4: Non-diabetic/CRS imipramine-treated group. Lane 5: Control diabetic group. Lane 6: Diabetic/CRS vehicle-treated group. Lane 7: Diabetic/CRS fluoxetine-treated group. Lane 8: Diabetic/CRS imipramine-treated group. (B) Effects of fluoxetine (FLU) versus imipramine (IMIP) on aortic TLR-4 gene expression (mRNA) as % normalized by β-actin quantity using semi-quantitative RT-PCR in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM (n = 6). ***P
Figure Legend Snippet: (A) Ethidium bromide-stained agarose gel electrophoresis showing the amplified RT-PCR products of TLR-4 (299bp) and β-actin (180 bp) as an internal standard, from aortic homogenates of Wistar rats. First Lane (MW): molecular weight ladder standard. Lane 1: control non-diabetic group. Lane 2: Non-diabetic/CRS vehicle-treated group. Lane 3: Non-diabetic/CRS fluoxetine-treated group. Lane 4: Non-diabetic/CRS imipramine-treated group. Lane 5: Control diabetic group. Lane 6: Diabetic/CRS vehicle-treated group. Lane 7: Diabetic/CRS fluoxetine-treated group. Lane 8: Diabetic/CRS imipramine-treated group. (B) Effects of fluoxetine (FLU) versus imipramine (IMIP) on aortic TLR-4 gene expression (mRNA) as % normalized by β-actin quantity using semi-quantitative RT-PCR in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM (n = 6). ***P

Techniques Used: Staining, Agarose Gel Electrophoresis, Amplification, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Expressing, Quantitative RT-PCR

Effects of fluoxetine (FLU) and imipramine (IMIP) on forced swimming (A) and open field (B) tests behavioral changes in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM of 9–14 rats/group. ***P
Figure Legend Snippet: Effects of fluoxetine (FLU) and imipramine (IMIP) on forced swimming (A) and open field (B) tests behavioral changes in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM of 9–14 rats/group. ***P

Techniques Used:

Photomicrographs of aortic sections stained by H E (X640) in different experimental groups. (A) Control non-diabetic group with unremarkable change, groups (B) Non-diabetic/CRS vehicle-treated group, (C) Non-diabetic/CRS fluoxetine-treated group (D) Non-diabetic/CRS imipramine-treated group, (E) Control diabetic group (F) Diabetic/CRS vehicle-treated group, (G) Diabetic/CRS fluoxetine-treated group, (H) Diabetic/CRS imipramine-treated group. Endothelial lining of tunica intima (↑) and vacuolation of cytoplasm of some smooth muscle fibers (▲).
Figure Legend Snippet: Photomicrographs of aortic sections stained by H E (X640) in different experimental groups. (A) Control non-diabetic group with unremarkable change, groups (B) Non-diabetic/CRS vehicle-treated group, (C) Non-diabetic/CRS fluoxetine-treated group (D) Non-diabetic/CRS imipramine-treated group, (E) Control diabetic group (F) Diabetic/CRS vehicle-treated group, (G) Diabetic/CRS fluoxetine-treated group, (H) Diabetic/CRS imipramine-treated group. Endothelial lining of tunica intima (↑) and vacuolation of cytoplasm of some smooth muscle fibers (▲).

Techniques Used: Staining

Effects of fluoxetine (FLU) versus imipramine (IMIP) on serum corticosterone in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM (n = 6). ***P
Figure Legend Snippet: Effects of fluoxetine (FLU) versus imipramine (IMIP) on serum corticosterone in diabetic and non-diabetic rats exposed to chronic restraint stress (CRS). Data are mean±SEM (n = 6). ***P

Techniques Used:

70) Product Images from "Female-Specific Flightless (fsRIDL) Phenotype for Control of Aedes albopictus"

Article Title: Female-Specific Flightless (fsRIDL) Phenotype for Control of Aedes albopictus

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001724

Characterisation of Ae. albopictus Actin-4 gene. A: Gene structure of Aedes albopictus and Aedes aegypti Actin-4 . Putative promoters are indicated by arrows, exons are shown as boxes, introns as lines. Non-coding 5′ and 3′UTR are shaded pale grey; the male-specific exons are shaded dark grey. B: RT-PCR confirming differential splicing in male (M) and female (F) Ae. albopictus pupae; genomic (g) and no template control (c) are also shown. L: DNA size marker (Smartladder, Eurogentec). Sizes of the major bands are consistent with the predicted gene structure (panel A and Table S1 ); sequence of the cDNA bands confirmed splice sites (not shown).
Figure Legend Snippet: Characterisation of Ae. albopictus Actin-4 gene. A: Gene structure of Aedes albopictus and Aedes aegypti Actin-4 . Putative promoters are indicated by arrows, exons are shown as boxes, introns as lines. Non-coding 5′ and 3′UTR are shaded pale grey; the male-specific exons are shaded dark grey. B: RT-PCR confirming differential splicing in male (M) and female (F) Ae. albopictus pupae; genomic (g) and no template control (c) are also shown. L: DNA size marker (Smartladder, Eurogentec). Sizes of the major bands are consistent with the predicted gene structure (panel A and Table S1 ); sequence of the cDNA bands confirmed splice sites (not shown).

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker, Sequencing

OX4358 structure, fluorescence phenotype and splicing. A: Map of the OX4358 construct. Promoters are indicated by arrows, exons are shown as boxes, introns as horizontal lines. The engineered start codon is indicated by a bar in the Actin-4 exon 1 (Ex1), whilst stop codons in the male exon are shown by bars below the line. B: Phenotype of wild-type (left) and OX4358F1-Aal (right) pupae under white light (left panel) and blue filter (right panel). C: RT-PCR analysis of transcripts from Ae. albopictus OX4358, from two male and two female pupae (M1, M2, F1 and F2, respectively), gDNA amplification (g) and no-template control (c). L: DNA size marker (Smartladder, Eurogentec). D: RT-PCR analysis of transcripts from Ae. aegypti OX4358, from three male and three female pupae (M1, M2, M3, F1, F2, and F3 respectively) and no template control (c). Sequencing of the PCR products revealed that splicing occurs as in the native gene, except for a second male-specific transcript in which exon 1 has an extra 75 bp in both Ae. aegypti and Ae. albopictus (see text).
Figure Legend Snippet: OX4358 structure, fluorescence phenotype and splicing. A: Map of the OX4358 construct. Promoters are indicated by arrows, exons are shown as boxes, introns as horizontal lines. The engineered start codon is indicated by a bar in the Actin-4 exon 1 (Ex1), whilst stop codons in the male exon are shown by bars below the line. B: Phenotype of wild-type (left) and OX4358F1-Aal (right) pupae under white light (left panel) and blue filter (right panel). C: RT-PCR analysis of transcripts from Ae. albopictus OX4358, from two male and two female pupae (M1, M2, F1 and F2, respectively), gDNA amplification (g) and no-template control (c). L: DNA size marker (Smartladder, Eurogentec). D: RT-PCR analysis of transcripts from Ae. aegypti OX4358, from three male and three female pupae (M1, M2, M3, F1, F2, and F3 respectively) and no template control (c). Sequencing of the PCR products revealed that splicing occurs as in the native gene, except for a second male-specific transcript in which exon 1 has an extra 75 bp in both Ae. aegypti and Ae. albopictus (see text).

Techniques Used: Fluorescence, Construct, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Sequencing, Polymerase Chain Reaction

71) Product Images from "Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels"

Article Title: Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2017.00232

Deletion of Cav1.4 decreases RS1 distribution in the retina. (A) The upper panel shows images taken at a lower magnification (20×) of WT (a1-a5) and Cav1.4 −/− (b1-b5) retinal sections that were double immunostained with antibodies against Cav1.4 and Ribeye. The lower panel shows images taken at a higher magnification (40×) of WT (c1-c5) and Cav1.4 −/− (d1-d5) retinal sections stained for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The upper panel shows images of WT retina stained with RS1 taken at lower (20×; a1-a3) and higher (40×; b1-b3) magnifications. Likewise, the lower panel shows images of Cav1.4 −/− retinal sections stained with RS1 at lower (20×; c1-c3) and higher (40×; d1-d3) magnifications. The scale bar represents 50 μm. DAPI stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.
Figure Legend Snippet: Deletion of Cav1.4 decreases RS1 distribution in the retina. (A) The upper panel shows images taken at a lower magnification (20×) of WT (a1-a5) and Cav1.4 −/− (b1-b5) retinal sections that were double immunostained with antibodies against Cav1.4 and Ribeye. The lower panel shows images taken at a higher magnification (40×) of WT (c1-c5) and Cav1.4 −/− (d1-d5) retinal sections stained for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The upper panel shows images of WT retina stained with RS1 taken at lower (20×; a1-a3) and higher (40×; b1-b3) magnifications. Likewise, the lower panel shows images of Cav1.4 −/− retinal sections stained with RS1 at lower (20×; c1-c3) and higher (40×; d1-d3) magnifications. The scale bar represents 50 μm. DAPI stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

Techniques Used: Staining

RS1 augments Cav1.4-LTCC in HEK cells. (A) Cells transfected with Cav1.4 subunit without other LTCC auxiliary subunits (Cav1.4 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.4 and RS1 do not elicit measurable LTCC currents carried by Ba 2+ ( I Ba ). (B) RS1 enhances the channel voltage-dependent activation (Norm. G) of functional Cav1.4-LTCCs. *Indicates a statistical difference between the two groups (* p
Figure Legend Snippet: RS1 augments Cav1.4-LTCC in HEK cells. (A) Cells transfected with Cav1.4 subunit without other LTCC auxiliary subunits (Cav1.4 + EGFP) do not have functional LTCCs. Co-transfection with Cav1.4 and RS1 do not elicit measurable LTCC currents carried by Ba 2+ ( I Ba ). (B) RS1 enhances the channel voltage-dependent activation (Norm. G) of functional Cav1.4-LTCCs. *Indicates a statistical difference between the two groups (* p

Techniques Used: Transfection, Functional Assay, Cotransfection, Activation Assay

Deletion of RS1 decreases the protein expression of Cav1.3 and Cav1.4. (A) The upper panel (a1-c5) contains retinal sections of wild type (WT), and the lower panel (d1-f5) contains retinal sections of RS1 −/− . (a1-a2) and (d1-d2) are the immunostaining for RS1. (b1-b5) and (e1-e5) are the double immunostaining for Cav1.3 and Ribeye; (c1-c5) and (f1-f5) are the double immunostaining for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The same immunostained retinal sections are shown at a higher magnification (40×). The upper panel contains retinal sections from WT (a1-a5) and RS1 −/− (b1-b5) that were double-stained for Cav1.3 and Ribeye. Images in (a1-a5) and (b1-b5) include retinal layers of IS, ONL, OPL and INL. The lower panel contains retinal sections from WT (c1-c5) and RS1 −/− (d1-d5) that were double-stained for Cav1.4 and Ribeye. Images in (c1-c5) and (d1-d5) include retinal layers of ONL, OPL and INL. The scale bar represents 50 μm. 4′ s ,6-diamidino-2-phenylindole (DAPI) stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.
Figure Legend Snippet: Deletion of RS1 decreases the protein expression of Cav1.3 and Cav1.4. (A) The upper panel (a1-c5) contains retinal sections of wild type (WT), and the lower panel (d1-f5) contains retinal sections of RS1 −/− . (a1-a2) and (d1-d2) are the immunostaining for RS1. (b1-b5) and (e1-e5) are the double immunostaining for Cav1.3 and Ribeye; (c1-c5) and (f1-f5) are the double immunostaining for Cav1.4 and Ribeye. The scale bar represents 50 μm. (B) The same immunostained retinal sections are shown at a higher magnification (40×). The upper panel contains retinal sections from WT (a1-a5) and RS1 −/− (b1-b5) that were double-stained for Cav1.3 and Ribeye. Images in (a1-a5) and (b1-b5) include retinal layers of IS, ONL, OPL and INL. The lower panel contains retinal sections from WT (c1-c5) and RS1 −/− (d1-d5) that were double-stained for Cav1.4 and Ribeye. Images in (c1-c5) and (d1-d5) include retinal layers of ONL, OPL and INL. The scale bar represents 50 μm. 4′ s ,6-diamidino-2-phenylindole (DAPI) stains the nuclei. BF, bright field; IS, photoreceptor inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer.

Techniques Used: Expressing, Immunostaining, Double Immunostaining, Staining

There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p
Figure Legend Snippet: There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p

Techniques Used: Luciferase, Transfection

72) Product Images from "Female-Specific Flightless (fsRIDL) Phenotype for Control of Aedes albopictus"

Article Title: Female-Specific Flightless (fsRIDL) Phenotype for Control of Aedes albopictus

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0001724

OX4358 structure, fluorescence phenotype and splicing. A: Map of the OX4358 construct. Promoters are indicated by arrows, exons are shown as boxes, introns as horizontal lines. The engineered start codon is indicated by a bar in the Actin-4 exon 1 (Ex1), whilst stop codons in the male exon are shown by bars below the line. B: Phenotype of wild-type (left) and OX4358F1-Aal (right) pupae under white light (left panel) and blue filter (right panel). C: RT-PCR analysis of transcripts from Ae. albopictus OX4358, from two male and two female pupae (M1, M2, F1 and F2, respectively), gDNA amplification (g) and no-template control (c). L: DNA size marker (Smartladder, Eurogentec). D: RT-PCR analysis of transcripts from Ae. aegypti OX4358, from three male and three female pupae (M1, M2, M3, F1, F2, and F3 respectively) and no template control (c). Sequencing of the PCR products revealed that splicing occurs as in the native gene, except for a second male-specific transcript in which exon 1 has an extra 75 bp in both Ae. aegypti and Ae. albopictus (see text).
Figure Legend Snippet: OX4358 structure, fluorescence phenotype and splicing. A: Map of the OX4358 construct. Promoters are indicated by arrows, exons are shown as boxes, introns as horizontal lines. The engineered start codon is indicated by a bar in the Actin-4 exon 1 (Ex1), whilst stop codons in the male exon are shown by bars below the line. B: Phenotype of wild-type (left) and OX4358F1-Aal (right) pupae under white light (left panel) and blue filter (right panel). C: RT-PCR analysis of transcripts from Ae. albopictus OX4358, from two male and two female pupae (M1, M2, F1 and F2, respectively), gDNA amplification (g) and no-template control (c). L: DNA size marker (Smartladder, Eurogentec). D: RT-PCR analysis of transcripts from Ae. aegypti OX4358, from three male and three female pupae (M1, M2, M3, F1, F2, and F3 respectively) and no template control (c). Sequencing of the PCR products revealed that splicing occurs as in the native gene, except for a second male-specific transcript in which exon 1 has an extra 75 bp in both Ae. aegypti and Ae. albopictus (see text).

Techniques Used: Fluorescence, Construct, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Sequencing, Polymerase Chain Reaction

73) Product Images from "Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps"

Article Title: Efficient conditional and promoter-specific in vivo expression of cDNAs of choice by taking advantage of recombinase-mediated cassette exchange using FlEx gene traps

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq044

PCR analysis of SB100 transfected exchange clone to determine mobilization of IR/DR flanked cassette. Genomic DNA of pooled SB100 transfected dishes (0, 10 and 70-µg SB100 plasmid) of exchange clone A03 (E307D01/pExFLP-dsRed derivative) was screened with primers located 5′ of the F3 site (B045), in the pA of dsRed (B048) and in the hygromycin-resistance cassette (H). DNA was screened with either primer combination H/B045 or B048/B045, as controls genomic DNA of clone A03 (A03 −SB) and E307D01 was used. Primers H/B045 yielded a 1-kb product after successful excision of the IR/DR flanked cassette. Without mobilization, a 839-bp band was amplified by primers B048/B045, whereas the original gene trap insertion led to a 631-bp band.
Figure Legend Snippet: PCR analysis of SB100 transfected exchange clone to determine mobilization of IR/DR flanked cassette. Genomic DNA of pooled SB100 transfected dishes (0, 10 and 70-µg SB100 plasmid) of exchange clone A03 (E307D01/pExFLP-dsRed derivative) was screened with primers located 5′ of the F3 site (B045), in the pA of dsRed (B048) and in the hygromycin-resistance cassette (H). DNA was screened with either primer combination H/B045 or B048/B045, as controls genomic DNA of clone A03 (A03 −SB) and E307D01 was used. Primers H/B045 yielded a 1-kb product after successful excision of the IR/DR flanked cassette. Without mobilization, a 839-bp band was amplified by primers B048/B045, whereas the original gene trap insertion led to a 631-bp band.

Techniques Used: Polymerase Chain Reaction, Transfection, Plasmid Preparation, Amplification

74) Product Images from "miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation"

Article Title: miR-22 Is a Novel Mediator of Vascular Smooth Muscle Cell Phenotypic Modulation and Neointima Formation

Journal: Circulation

doi: 10.1161/CIRCULATIONAHA.117.027799

EVI1 is the novel target of miR-22 in VSMCs. A , The predicted miR-22 binding site within EVI1 3′-UTR by Targetscan. EVI1, miR-22 sequence (mmu-miR-22), and the miR-22 binding site mutant (EVI1 mutant) are depicted in this illustration. The mutation site in EVI1 mutant and corresponding sequences in wild-type EVI1 and mmu-miR-22 are underlined and bold. B , EVI1 was upregulated in the extended cultured VSMCs. Each dot represents EVI1 mRNA level in each passage (P3, P8, P9, P12) normalized to EVI1 mRNA level of P0. C and D , EVI1 was negatively regulated by miR-22. VSMCs transfected with miR-22 mimics, inhibitor, or respective controls (Ctrl), as indicated, were subjected to serum starvation for 48 hours. Total RNA and protein were harvested and subjected to RT-qPCR ( C ) and Western blot ( D ) analyses, respectively. E , miR-22 repressed EVI1 3′-UTR reporter activity. miR-22 mimics, inhibitor, or respective controls (Ctrl) were cotransfected with EVI1 3′-UTR reporter into VSMCs, as indicated. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. F , miR-22 binding site was required for miR-22–mediated EVI1 gene repression. miR-22 mimics or control miR mimics (miRNA ctrl) were cotransfected into VSMCs with wild-type reporter (pmiR-EVI1-WT) or the reporter containing mutated miR-22 binding site (pmiR-EVI1-mutant). Transfected cells were subjected to serum starvation for 48 hours before luciferase activity assay. Data and error bars in B through F are representative ( D ) or mean±SEM ( B , C , E , and F ) (n=4 in D and F ). * P
Figure Legend Snippet: EVI1 is the novel target of miR-22 in VSMCs. A , The predicted miR-22 binding site within EVI1 3′-UTR by Targetscan. EVI1, miR-22 sequence (mmu-miR-22), and the miR-22 binding site mutant (EVI1 mutant) are depicted in this illustration. The mutation site in EVI1 mutant and corresponding sequences in wild-type EVI1 and mmu-miR-22 are underlined and bold. B , EVI1 was upregulated in the extended cultured VSMCs. Each dot represents EVI1 mRNA level in each passage (P3, P8, P9, P12) normalized to EVI1 mRNA level of P0. C and D , EVI1 was negatively regulated by miR-22. VSMCs transfected with miR-22 mimics, inhibitor, or respective controls (Ctrl), as indicated, were subjected to serum starvation for 48 hours. Total RNA and protein were harvested and subjected to RT-qPCR ( C ) and Western blot ( D ) analyses, respectively. E , miR-22 repressed EVI1 3′-UTR reporter activity. miR-22 mimics, inhibitor, or respective controls (Ctrl) were cotransfected with EVI1 3′-UTR reporter into VSMCs, as indicated. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. F , miR-22 binding site was required for miR-22–mediated EVI1 gene repression. miR-22 mimics or control miR mimics (miRNA ctrl) were cotransfected into VSMCs with wild-type reporter (pmiR-EVI1-WT) or the reporter containing mutated miR-22 binding site (pmiR-EVI1-mutant). Transfected cells were subjected to serum starvation for 48 hours before luciferase activity assay. Data and error bars in B through F are representative ( D ) or mean±SEM ( B , C , E , and F ) (n=4 in D and F ). * P

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Cell Culture, Transfection, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase

EVI1 inhibition reproduces the effects of miR-22 overexpression on VSMC-specific gene expression, proliferation, and migration. A and B , EVI1 knockdown VSMC was generated and validated. Total RNA and protein of control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs were harvested and subjected to RT-qPCR ( A ) and Western blot ( B ) analyses, respectively. C , EVI1 knockdown significantly increases expression of VSMC markers (SMαA, SM22α, CNN1, SM-myh11, SMTN-B) and transcription factors (SRF and Myocd), although the transcription factor MEF2c exhibited no significant change in expression. Total RNA of control and EVI1 stable knockdown VSMCs were harvested and subjected to RT-qPCR. D , Inhibition of endogenous EVI1 decreases VSMC proliferation. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by BrdU incorporation assays in response to no (Ctrl), serum (20%), and PDGF-BB (10 ng/mL) stimulation. E , Inhibition of endogenous EVI1 decreases VSMC migration. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by transwell migration in response to serum (20%) and PDGF-BB (30 ng/mL) stimulation. Note: No or very few migrated cells were observed without cell chemoattractant in transwell migration assays; therefore, no control treatment is shown. Data and error bars are representative ( B ) or mean±SEM ( A and C through E ) (n=3 in C ; 4 in D ; or 5 in E ). * P
Figure Legend Snippet: EVI1 inhibition reproduces the effects of miR-22 overexpression on VSMC-specific gene expression, proliferation, and migration. A and B , EVI1 knockdown VSMC was generated and validated. Total RNA and protein of control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs were harvested and subjected to RT-qPCR ( A ) and Western blot ( B ) analyses, respectively. C , EVI1 knockdown significantly increases expression of VSMC markers (SMαA, SM22α, CNN1, SM-myh11, SMTN-B) and transcription factors (SRF and Myocd), although the transcription factor MEF2c exhibited no significant change in expression. Total RNA of control and EVI1 stable knockdown VSMCs were harvested and subjected to RT-qPCR. D , Inhibition of endogenous EVI1 decreases VSMC proliferation. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by BrdU incorporation assays in response to no (Ctrl), serum (20%), and PDGF-BB (10 ng/mL) stimulation. E , Inhibition of endogenous EVI1 decreases VSMC migration. Control and EVI1 stable knockdown VSMCs were subjected to serum starvation for 48 hours, followed by transwell migration in response to serum (20%) and PDGF-BB (30 ng/mL) stimulation. Note: No or very few migrated cells were observed without cell chemoattractant in transwell migration assays; therefore, no control treatment is shown. Data and error bars are representative ( B ) or mean±SEM ( A and C through E ) (n=3 in C ; 4 in D ; or 5 in E ). * P

Techniques Used: Inhibition, Over Expression, Expressing, Migration, Generated, shRNA, Quantitative RT-PCR, Western Blot, BrdU Incorporation Assay

EVI1 functions as a transcriptional repressor for VSMC gene expression. A and B , SRF binding element(s) are required for EVI1-mediated SMαA and SM22α gene promoter activity. Wild-type (SMαA/SM22α) or SRF binding site(s) mutants (SRF mut ) of SMαA ( A ) and SM22α ( B ) gene promoter reporters were transfected into control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. C and D , EVI1 inhibition significantly increases SRF and Myocd gene promoter activity. SRF ( C ) and Myocd ( D ) gene promoter reporters were transfected into control and EVI1 stable knockdown VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. E , EVI1 was significantly enriched at the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes and was significantly decreased by EVI1 knockdown, indicating direct binding of EVI1 to these promoter regions. ChIP assays were performed to measure EVI1 enrichment in the promoter region of its downstream targets. Control (sh-EVI1–) and EVI1 stable knockdown (sh-EVI1+) VSMCs were lysed and incubated with antibody against EVI1 to immunoprecipitate EVI1-bound promoter DNA, followed by qPCR to quantify DNA enrichment. For SMαA and SM22α, genomic DNA without SRF binding sites were amplified as additional control for specific promoter DNA enrichment and designated as Promoter–. For SRF and Myocd, PCR amplification of DNA region adjacent to their promoter was designated as Promoter–. F , H3K9me3 enrichment within the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes was significantly decreased by EVI1 knockdown. ChIP assays were performed by using an antibody against H3K9me3. Data and error bars are mean±SEM. * P
Figure Legend Snippet: EVI1 functions as a transcriptional repressor for VSMC gene expression. A and B , SRF binding element(s) are required for EVI1-mediated SMαA and SM22α gene promoter activity. Wild-type (SMαA/SM22α) or SRF binding site(s) mutants (SRF mut ) of SMαA ( A ) and SM22α ( B ) gene promoter reporters were transfected into control (nontarget shRNA, sh-NT) and EVI1 stable knockdown (EVI1 shRNA, sh-EVI1) VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. C and D , EVI1 inhibition significantly increases SRF and Myocd gene promoter activity. SRF ( C ) and Myocd ( D ) gene promoter reporters were transfected into control and EVI1 stable knockdown VSMCs. Transfected cells were subjected to serum starvation for 48 hours, and cell lysates were subjected to luciferase activity assay. E , EVI1 was significantly enriched at the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes and was significantly decreased by EVI1 knockdown, indicating direct binding of EVI1 to these promoter regions. ChIP assays were performed to measure EVI1 enrichment in the promoter region of its downstream targets. Control (sh-EVI1–) and EVI1 stable knockdown (sh-EVI1+) VSMCs were lysed and incubated with antibody against EVI1 to immunoprecipitate EVI1-bound promoter DNA, followed by qPCR to quantify DNA enrichment. For SMαA and SM22α, genomic DNA without SRF binding sites were amplified as additional control for specific promoter DNA enrichment and designated as Promoter–. For SRF and Myocd, PCR amplification of DNA region adjacent to their promoter was designated as Promoter–. F , H3K9me3 enrichment within the promoter regions of SMαA ( far left ), SM22α ( middle left ), SRF ( middle right ), and Myocd ( far right ) genes was significantly decreased by EVI1 knockdown. ChIP assays were performed by using an antibody against H3K9me3. Data and error bars are mean±SEM. * P

Techniques Used: Expressing, Binding Assay, Activity Assay, Transfection, shRNA, Luciferase, Inhibition, Chromatin Immunoprecipitation, Incubation, Real-time Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

75) Product Images from "Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels"

Article Title: Retinoschisin Facilitates the Function of L-Type Voltage-Gated Calcium Channels

Journal: Frontiers in Cellular Neuroscience

doi: 10.3389/fncel.2017.00232

There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p
Figure Legend Snippet: There is a physical interaction between retinoschisin (RS1) and L-type voltage-gated calcium channel (LTCC)α1 subunits. (A) Anti-RS1 antibody (RS1 Ab) is able to co-immunoprecipitate Cav1.3 from the porcine retina. (B) RS1 Ab is able to co-immunoprecipitate Cav1.4 from the porcine retina. (C) The whole cell lysates as loading control for (A,B) . (D) Mammalian two-hybrid (luciferase reporter) assays show that hRS1 is able to interact with the first 500 amino acids from the N-terminus of Cav1.4 (hCav1.4-N) including the first motif (I). Cells co-transfected with hRS1 and hCav1.4-N (hRS1 + hCav1.4-N) have significantly higher luciferase activities than the other two control groups ( n = 6 for each group, * p

Techniques Used: Luciferase, Transfection

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Polymerase Chain Reaction:

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Quantitative RT-PCR:

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In Silico:

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Expressing:

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Chloramphenicol Acetyltransferase Assay:

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression
Article Snippet: .. Next, cDNA was amplified (38 cycles of 95 °C for 20 s, 62 °C for 15 s, and 72 °C for 20 s) with SYBR qPCR SuperMix (Novoprotein, Shanghai, China) according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse Foxo1 gene, 5′ – TGT TTG ATT CAT TTC CTT TGG T -3′ and 5′ –TGA TTT TCT CCG CTT ACT GTT G -3′; mouse CD127 gene, 5′-AAA AGT AAA GCA TGA TGT GGC C -3′ and 5′ –TTG AAG TAA TCG TTA TGG GGA A -3′; mouse Icos gene, 5′ – CAT TCC CAA CAC GAA CAC CTA A -3′ and 5′ –TCT TCA CCC CCA GAA AAC ACA G -3′; mouse Aven gene, 5′- GGG ACC AGG AAC CAG AAA AAG A -3′ and 5′ – TAC ACA GAA GGC AAC CAG CAT T -3′; mouse IL-2 gene, 5′- GAT GAA CTT GGA CCT CTG CG -3′ and 5′- AGG GCT TGT TGA GAT GAT GC -3′; mouse IL-4 gene, 5′- CAT CCT GCT CTT CTT TCT CG -3′ and 5′- CCT TCT CCT GTG ACC TCG TT -3′; mouse IL-7 gene, 5′- GTT ATG GCA AAG CCA GAG CG -3′ and 5′- TGC GGG AGG TGG GTG TAG TC-3′; mouse IL-14 gene, 5′- CCC CTT CTG TCC AGC CAC TC -3′ and 5′- TCC CGT CTT CG TCC AA TCT-3′. mouse Bcl2 gene, 5′-GC TAC CGT CGT GAC TTC GC -3′ and 5′- ATC CCA GCC TCC GTT ATC C-3′. .. For each gene, the mRNA level was normalized against GAPDH expression in the respective cDNA preparation.

Countercurrent Chromatography:

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression
Article Snippet: .. Next, cDNA was amplified (38 cycles of 95 °C for 20 s, 62 °C for 15 s, and 72 °C for 20 s) with SYBR qPCR SuperMix (Novoprotein, Shanghai, China) according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse Foxo1 gene, 5′ – TGT TTG ATT CAT TTC CTT TGG T -3′ and 5′ –TGA TTT TCT CCG CTT ACT GTT G -3′; mouse CD127 gene, 5′-AAA AGT AAA GCA TGA TGT GGC C -3′ and 5′ –TTG AAG TAA TCG TTA TGG GGA A -3′; mouse Icos gene, 5′ – CAT TCC CAA CAC GAA CAC CTA A -3′ and 5′ –TCT TCA CCC CCA GAA AAC ACA G -3′; mouse Aven gene, 5′- GGG ACC AGG AAC CAG AAA AAG A -3′ and 5′ – TAC ACA GAA GGC AAC CAG CAT T -3′; mouse IL-2 gene, 5′- GAT GAA CTT GGA CCT CTG CG -3′ and 5′- AGG GCT TGT TGA GAT GAT GC -3′; mouse IL-4 gene, 5′- CAT CCT GCT CTT CTT TCT CG -3′ and 5′- CCT TCT CCT GTG ACC TCG TT -3′; mouse IL-7 gene, 5′- GTT ATG GCA AAG CCA GAG CG -3′ and 5′- TGC GGG AGG TGG GTG TAG TC-3′; mouse IL-14 gene, 5′- CCC CTT CTG TCC AGC CAC TC -3′ and 5′- TCC CGT CTT CG TCC AA TCT-3′. mouse Bcl2 gene, 5′-GC TAC CGT CGT GAC TTC GC -3′ and 5′- ATC CCA GCC TCC GTT ATC C-3′. .. For each gene, the mRNA level was normalized against GAPDH expression in the respective cDNA preparation.

Cell Differentiation:

Article Title: Methylation of Gata3 Protein at Arg-261 Regulates Transactivation of the Il5 Gene in T Helper 2 Cells *
Article Snippet: Kimura M., Koseki Y., Yamashita M., Watanabe N., Shimizu C., Katsumoto T., Kitamura T., Taniguchi M., Koseki H., Nakayama T. (2001) Regulation of Th2 cell differentiation by mel-18 , a mammalian polycomb group gene . .. Omori M., Yamashita M., Inami M., Ukai-Tadenuma M., Kimura M., Nigo Y., Hosokawa H., Hasegawa A., Taniguchi M., Nakayama T. (2003) CD8 T cell-specific downregulation of histone hyperacetylation and gene activation of the IL-4 gene locus by ROG, repressor of GATA .

other:

Article Title: Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells
Article Snippet: The transcripts of Mcp-2 and Mcp-4 but not Mcp-5 contain multiple UGXCCCC motifs in their 3′-UTRs, and these potential cis -acting elements are thought to be responsible for the reduced stability of the Mcp-2 and Mcp-4 transcript , .

Article Title: TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival
Article Snippet: In fact, MMP-2 and MMP-9 have been reported to have an important role in invasion and metastasis of glioma and other cancers., and

Article Title: Generation of a New Congenic Mouse Strain with Enhanced Chymase Expression in Mast Cells
Article Snippet: Mcp-2 and Mcp-4 both have expected molecular sizes of about 27 kDa and are most relevant to mast cells.

Article Title: TLX activates MMP-2, promotes self-renewal of tumor spheres in neuroblastoma and correlates with poor patient survival
Article Snippet: Ki67 (SC23900), Oct-4 (SC8630), MMP-2 (SC53630) and HIF-2α (SC46691) were also used.

Sequencing:

Article Title: Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs
Article Snippet: .. PCR carried out with the same primers on the greater hedgehog tenrec ( S. setosus ) DNA resulted in the amplification of the orthologous fragment , with a full-length env gene ORF ( ) followed by a 3′ LTR and a 600-bp flanking sequence with ∼95% nucleotide identity to that of E . telfairi , confirming the conservation of ten-env1 at the orthologous genomic locus in E . telfairi and S. setosus . ..

Article Title: Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes
Article Snippet: Methods: We designed four new primer pairs to amplify mcr-1 , mcr-2 , mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. .. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data.

Injection:

Article Title: Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen
Article Snippet: HDM exposure induces the recruitment of IL-4–competent basophils and eosinophils to the draining LNs The use of sensitive transgenic IL-4 reporter mice has greatly advanced our understanding of innate type 2 immunity, as eosinophils, mast cells, and basophils were found to have an accessible IL-4 gene locus, thus providing a surrogate marker for identification of these cells in vivo when IL4 -enhanced GFP (eGFP) is combined with lineage defining specific antibodies ( ; ). .. At 3 d after injection, a population of non-T, non-B (CD3− CD19− ) eGFP+ cells was observed in the MLNs of mice injected with HDM, whereas this population was not seen when PBS was injected.

Cellular Antioxidant Activity Assay:

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression
Article Snippet: .. Next, cDNA was amplified (38 cycles of 95 °C for 20 s, 62 °C for 15 s, and 72 °C for 20 s) with SYBR qPCR SuperMix (Novoprotein, Shanghai, China) according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse Foxo1 gene, 5′ – TGT TTG ATT CAT TTC CTT TGG T -3′ and 5′ –TGA TTT TCT CCG CTT ACT GTT G -3′; mouse CD127 gene, 5′-AAA AGT AAA GCA TGA TGT GGC C -3′ and 5′ –TTG AAG TAA TCG TTA TGG GGA A -3′; mouse Icos gene, 5′ – CAT TCC CAA CAC GAA CAC CTA A -3′ and 5′ –TCT TCA CCC CCA GAA AAC ACA G -3′; mouse Aven gene, 5′- GGG ACC AGG AAC CAG AAA AAG A -3′ and 5′ – TAC ACA GAA GGC AAC CAG CAT T -3′; mouse IL-2 gene, 5′- GAT GAA CTT GGA CCT CTG CG -3′ and 5′- AGG GCT TGT TGA GAT GAT GC -3′; mouse IL-4 gene, 5′- CAT CCT GCT CTT CTT TCT CG -3′ and 5′- CCT TCT CCT GTG ACC TCG TT -3′; mouse IL-7 gene, 5′- GTT ATG GCA AAG CCA GAG CG -3′ and 5′- TGC GGG AGG TGG GTG TAG TC-3′; mouse IL-14 gene, 5′- CCC CTT CTG TCC AGC CAC TC -3′ and 5′- TCC CGT CTT CG TCC AA TCT-3′. mouse Bcl2 gene, 5′-GC TAC CGT CGT GAC TTC GC -3′ and 5′- ATC CCA GCC TCC GTT ATC C-3′. .. For each gene, the mRNA level was normalized against GAPDH expression in the respective cDNA preparation.

Marker:

Article Title: Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen
Article Snippet: .. HDM exposure induces the recruitment of IL-4–competent basophils and eosinophils to the draining LNs The use of sensitive transgenic IL-4 reporter mice has greatly advanced our understanding of innate type 2 immunity, as eosinophils, mast cells, and basophils were found to have an accessible IL-4 gene locus, thus providing a surrogate marker for identification of these cells in vivo when IL4 -enhanced GFP (eGFP) is combined with lineage defining specific antibodies ( ; ). .. We therefore examined the appearance of eGFP+ cells in the lung draining MLNs of BALB/c 4get mice (IL-4 GFP-enhanced transcript) that received a single intranasal (i.n.) administration of an extract of the clinically relevant allergen HDM.

In Vivo:

Article Title: Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen
Article Snippet: .. HDM exposure induces the recruitment of IL-4–competent basophils and eosinophils to the draining LNs The use of sensitive transgenic IL-4 reporter mice has greatly advanced our understanding of innate type 2 immunity, as eosinophils, mast cells, and basophils were found to have an accessible IL-4 gene locus, thus providing a surrogate marker for identification of these cells in vivo when IL4 -enhanced GFP (eGFP) is combined with lineage defining specific antibodies ( ; ). .. We therefore examined the appearance of eGFP+ cells in the lung draining MLNs of BALB/c 4get mice (IL-4 GFP-enhanced transcript) that received a single intranasal (i.n.) administration of an extract of the clinically relevant allergen HDM.

Methylation:

Article Title: Methylation of Gata3 Protein at Arg-261 Regulates Transactivation of the Il5 Gene in T Helper 2 Cells *
Article Snippet: Omori M., Yamashita M., Inami M., Ukai-Tadenuma M., Kimura M., Nigo Y., Hosokawa H., Hasegawa A., Taniguchi M., Nakayama T. (2003) CD8 T cell-specific downregulation of histone hyperacetylation and gene activation of the IL-4 gene locus by ROG, repressor of GATA . .. Campbell M., Chang P. C., Huerta S., Izumiya C., Davis R., Tepper C. G., Kim K. Y., Shevchenko B., Wang D. H., Jung J. U., Luciw P. A., Kung H. J., Izumiya Y. (2012) Protein arginine methyltransferase 1-directed methylation of Kaposi sarcoma-associated herpesvirus latency-associated nuclear antigen .

Mouse Assay:

Article Title: Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen
Article Snippet: .. HDM exposure induces the recruitment of IL-4–competent basophils and eosinophils to the draining LNs The use of sensitive transgenic IL-4 reporter mice has greatly advanced our understanding of innate type 2 immunity, as eosinophils, mast cells, and basophils were found to have an accessible IL-4 gene locus, thus providing a surrogate marker for identification of these cells in vivo when IL4 -enhanced GFP (eGFP) is combined with lineage defining specific antibodies ( ; ). .. We therefore examined the appearance of eGFP+ cells in the lung draining MLNs of BALB/c 4get mice (IL-4 GFP-enhanced transcript) that received a single intranasal (i.n.) administration of an extract of the clinically relevant allergen HDM.

Article Title: Batf is important for IL-4 expression in T follicular helper cells
Article Snippet: Batf in the complex with IRF4 and along with Stat3 and Stat6 binds to the CNS2 region in the IL-4 gene locus and facilitates IL-4 transcription in Tfh cells. .. Specifically, deletion of Batf in Tfh cells protects mice from induction of allergic asthma; Batf may be targeted in treating allergic inflammatory diseases.

Transgenic Assay:

Article Title: Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen
Article Snippet: .. HDM exposure induces the recruitment of IL-4–competent basophils and eosinophils to the draining LNs The use of sensitive transgenic IL-4 reporter mice has greatly advanced our understanding of innate type 2 immunity, as eosinophils, mast cells, and basophils were found to have an accessible IL-4 gene locus, thus providing a surrogate marker for identification of these cells in vivo when IL4 -enhanced GFP (eGFP) is combined with lineage defining specific antibodies ( ; ). .. We therefore examined the appearance of eGFP+ cells in the lung draining MLNs of BALB/c 4get mice (IL-4 GFP-enhanced transcript) that received a single intranasal (i.n.) administration of an extract of the clinically relevant allergen HDM.

Activated Clotting Time Assay:

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression
Article Snippet: .. Next, cDNA was amplified (38 cycles of 95 °C for 20 s, 62 °C for 15 s, and 72 °C for 20 s) with SYBR qPCR SuperMix (Novoprotein, Shanghai, China) according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse Foxo1 gene, 5′ – TGT TTG ATT CAT TTC CTT TGG T -3′ and 5′ –TGA TTT TCT CCG CTT ACT GTT G -3′; mouse CD127 gene, 5′-AAA AGT AAA GCA TGA TGT GGC C -3′ and 5′ –TTG AAG TAA TCG TTA TGG GGA A -3′; mouse Icos gene, 5′ – CAT TCC CAA CAC GAA CAC CTA A -3′ and 5′ –TCT TCA CCC CCA GAA AAC ACA G -3′; mouse Aven gene, 5′- GGG ACC AGG AAC CAG AAA AAG A -3′ and 5′ – TAC ACA GAA GGC AAC CAG CAT T -3′; mouse IL-2 gene, 5′- GAT GAA CTT GGA CCT CTG CG -3′ and 5′- AGG GCT TGT TGA GAT GAT GC -3′; mouse IL-4 gene, 5′- CAT CCT GCT CTT CTT TCT CG -3′ and 5′- CCT TCT CCT GTG ACC TCG TT -3′; mouse IL-7 gene, 5′- GTT ATG GCA AAG CCA GAG CG -3′ and 5′- TGC GGG AGG TGG GTG TAG TC-3′; mouse IL-14 gene, 5′- CCC CTT CTG TCC AGC CAC TC -3′ and 5′- TCC CGT CTT CG TCC AA TCT-3′. mouse Bcl2 gene, 5′-GC TAC CGT CGT GAC TTC GC -3′ and 5′- ATC CCA GCC TCC GTT ATC C-3′. .. For each gene, the mRNA level was normalized against GAPDH expression in the respective cDNA preparation.

Plasmid Preparation:

Article Title: Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes
Article Snippet: Plasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. .. Methods: We designed four new primer pairs to amplify mcr-1 , mcr-2 , mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons.

Real-time Polymerase Chain Reaction:

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression
Article Snippet: .. Next, cDNA was amplified (38 cycles of 95 °C for 20 s, 62 °C for 15 s, and 72 °C for 20 s) with SYBR qPCR SuperMix (Novoprotein, Shanghai, China) according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse Foxo1 gene, 5′ – TGT TTG ATT CAT TTC CTT TGG T -3′ and 5′ –TGA TTT TCT CCG CTT ACT GTT G -3′; mouse CD127 gene, 5′-AAA AGT AAA GCA TGA TGT GGC C -3′ and 5′ –TTG AAG TAA TCG TTA TGG GGA A -3′; mouse Icos gene, 5′ – CAT TCC CAA CAC GAA CAC CTA A -3′ and 5′ –TCT TCA CCC CCA GAA AAC ACA G -3′; mouse Aven gene, 5′- GGG ACC AGG AAC CAG AAA AAG A -3′ and 5′ – TAC ACA GAA GGC AAC CAG CAT T -3′; mouse IL-2 gene, 5′- GAT GAA CTT GGA CCT CTG CG -3′ and 5′- AGG GCT TGT TGA GAT GAT GC -3′; mouse IL-4 gene, 5′- CAT CCT GCT CTT CTT TCT CG -3′ and 5′- CCT TCT CCT GTG ACC TCG TT -3′; mouse IL-7 gene, 5′- GTT ATG GCA AAG CCA GAG CG -3′ and 5′- TGC GGG AGG TGG GTG TAG TC-3′; mouse IL-14 gene, 5′- CCC CTT CTG TCC AGC CAC TC -3′ and 5′- TCC CGT CTT CG TCC AA TCT-3′. mouse Bcl2 gene, 5′-GC TAC CGT CGT GAC TTC GC -3′ and 5′- ATC CCA GCC TCC GTT ATC C-3′. .. For each gene, the mRNA level was normalized against GAPDH expression in the respective cDNA preparation.

Multiplex Assay:

Article Title: Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes
Article Snippet: A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5 , and variants) in Enterobacteriaceae was developed for surveillance or research purposes. .. Methods: We designed four new primer pairs to amplify mcr-1 , mcr-2 , mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons.

Activation Assay:

Article Title: Methylation of Gata3 Protein at Arg-261 Regulates Transactivation of the Il5 Gene in T Helper 2 Cells *
Article Snippet: .. Omori M., Yamashita M., Inami M., Ukai-Tadenuma M., Kimura M., Nigo Y., Hosokawa H., Hasegawa A., Taniguchi M., Nakayama T. (2003) CD8 T cell-specific downregulation of histone hyperacetylation and gene activation of the IL-4 gene locus by ROG, repressor of GATA . ..

CTG Assay:

Article Title: Forkhead-box transcription factor 1 affects the apoptosis of natural regulatory T cells by controlling Aven expression
Article Snippet: .. Next, cDNA was amplified (38 cycles of 95 °C for 20 s, 62 °C for 15 s, and 72 °C for 20 s) with SYBR qPCR SuperMix (Novoprotein, Shanghai, China) according to the manufacturer’s instructions, using gene-specific sets of primers: f for mouse Foxo1 gene, 5′ – TGT TTG ATT CAT TTC CTT TGG T -3′ and 5′ –TGA TTT TCT CCG CTT ACT GTT G -3′; mouse CD127 gene, 5′-AAA AGT AAA GCA TGA TGT GGC C -3′ and 5′ –TTG AAG TAA TCG TTA TGG GGA A -3′; mouse Icos gene, 5′ – CAT TCC CAA CAC GAA CAC CTA A -3′ and 5′ –TCT TCA CCC CCA GAA AAC ACA G -3′; mouse Aven gene, 5′- GGG ACC AGG AAC CAG AAA AAG A -3′ and 5′ – TAC ACA GAA GGC AAC CAG CAT T -3′; mouse IL-2 gene, 5′- GAT GAA CTT GGA CCT CTG CG -3′ and 5′- AGG GCT TGT TGA GAT GAT GC -3′; mouse IL-4 gene, 5′- CAT CCT GCT CTT CTT TCT CG -3′ and 5′- CCT TCT CCT GTG ACC TCG TT -3′; mouse IL-7 gene, 5′- GTT ATG GCA AAG CCA GAG CG -3′ and 5′- TGC GGG AGG TGG GTG TAG TC-3′; mouse IL-14 gene, 5′- CCC CTT CTG TCC AGC CAC TC -3′ and 5′- TCC CGT CTT CG TCC AA TCT-3′. mouse Bcl2 gene, 5′-GC TAC CGT CGT GAC TTC GC -3′ and 5′- ATC CCA GCC TCC GTT ATC C-3′. .. For each gene, the mRNA level was normalized against GAPDH expression in the respective cDNA preparation.

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    4Gene amplification polymerase chain reaction pcr
    Amplification Polymerase Chain Reaction Pcr, supplied by 4Gene, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amplification polymerase chain reaction pcr/product/4Gene
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