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    Structured Review

    Roche p malariae strains greece
    Antibody competition titration assays using MSP1 19 proteins from four Plasmodium species. A combined dilution (1:400 of each serum) containing sera from chimpanzees experimentally infected with either P. <t>malariae</t> (Klimatis), P. ovale (Alpert) or P. vivax (Duff) was incubated with the indicated concentrations of the MSP1 19 competitor protein for 1 h at room temperature. Competitor proteins used were: a P. falciparum MSP1 19 ; b P. malariae MSP1 19 ; c P. ovale MSP1 19 ; d P. vivax MSP1 19 . Multiplex bead assays were performed as described in “ Methods ” and the multiplex response in MFI-bg units are plotted versus the competitor concentration. Multiplex responses are presented as a percentage of the assay results for the PBS control
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    1) Product Images from "Specificity of the IgG antibody response to Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale MSP119 subunit proteins in multiplexed serologic assays"

    Article Title: Specificity of the IgG antibody response to Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale MSP119 subunit proteins in multiplexed serologic assays

    Journal: Malaria Journal

    doi: 10.1186/s12936-018-2566-0

    Antibody competition titration assays using MSP1 19 proteins from four Plasmodium species. A combined dilution (1:400 of each serum) containing sera from chimpanzees experimentally infected with either P. malariae (Klimatis), P. ovale (Alpert) or P. vivax (Duff) was incubated with the indicated concentrations of the MSP1 19 competitor protein for 1 h at room temperature. Competitor proteins used were: a P. falciparum MSP1 19 ; b P. malariae MSP1 19 ; c P. ovale MSP1 19 ; d P. vivax MSP1 19 . Multiplex bead assays were performed as described in “ Methods ” and the multiplex response in MFI-bg units are plotted versus the competitor concentration. Multiplex responses are presented as a percentage of the assay results for the PBS control
    Figure Legend Snippet: Antibody competition titration assays using MSP1 19 proteins from four Plasmodium species. A combined dilution (1:400 of each serum) containing sera from chimpanzees experimentally infected with either P. malariae (Klimatis), P. ovale (Alpert) or P. vivax (Duff) was incubated with the indicated concentrations of the MSP1 19 competitor protein for 1 h at room temperature. Competitor proteins used were: a P. falciparum MSP1 19 ; b P. malariae MSP1 19 ; c P. ovale MSP1 19 ; d P. vivax MSP1 19 . Multiplex bead assays were performed as described in “ Methods ” and the multiplex response in MFI-bg units are plotted versus the competitor concentration. Multiplex responses are presented as a percentage of the assay results for the PBS control

    Techniques Used: Titration, Infection, Incubation, Multiplex Assay, Concentration Assay

    Antibody competition titration assays using homologous MSP1 19 proteins. Dilutions (1:400) of P. falciparum Lot 6 defined human serum or of sera from chimpanzees experimentally infected with either P. malariae (Klimatis), P. ovale (Alpert) or P. vivax (Duff) were incubated with the indicated concentrations of the homologous MSP1 19 competitor protein for 1 h at room temperature. Multiplex bead assays were performed as described in “ Methods ”, and the multiplex responses in MFI-bg units are plotted versus the competitor concentration
    Figure Legend Snippet: Antibody competition titration assays using homologous MSP1 19 proteins. Dilutions (1:400) of P. falciparum Lot 6 defined human serum or of sera from chimpanzees experimentally infected with either P. malariae (Klimatis), P. ovale (Alpert) or P. vivax (Duff) were incubated with the indicated concentrations of the homologous MSP1 19 competitor protein for 1 h at room temperature. Multiplex bead assays were performed as described in “ Methods ”, and the multiplex responses in MFI-bg units are plotted versus the competitor concentration

    Techniques Used: Titration, Infection, Incubation, Multiplex Assay, Concentration Assay

    Alignment of predicted Plasmodium spp. MSP1 19 protein sequences using COBALT [ 61 ]. Residues in the P. malariae sequence that differ from the Cameroon sequence of Birkenmeyer et al. [ 38 ] are shaded. Predicted protein sequences resulting from the oligonucleotides used in PCR amplification are underlined. The positions of residues conserved among all the presented MSP1 19 protein sequences are indicated in the consensus with divergent residues indicated by a dot. GenBank accession numbers are MH577181, P. ovale Nigeria I strain; MH577182, P. malariae China I strain; MH577183, P. malariae Greece I strain; MH577184, P. malariae Uganda I strain; and MH577185, P. malariae Guyana strain
    Figure Legend Snippet: Alignment of predicted Plasmodium spp. MSP1 19 protein sequences using COBALT [ 61 ]. Residues in the P. malariae sequence that differ from the Cameroon sequence of Birkenmeyer et al. [ 38 ] are shaded. Predicted protein sequences resulting from the oligonucleotides used in PCR amplification are underlined. The positions of residues conserved among all the presented MSP1 19 protein sequences are indicated in the consensus with divergent residues indicated by a dot. GenBank accession numbers are MH577181, P. ovale Nigeria I strain; MH577182, P. malariae China I strain; MH577183, P. malariae Greece I strain; MH577184, P. malariae Uganda I strain; and MH577185, P. malariae Guyana strain

    Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification

    2) Product Images from "The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity"

    Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31364-y

    Alternative splicing of SCN8A exon 18 in the neoplasia-carcinoma sequence of human cervical tissue. ( A ) Alternative splicing of SCN8A Exon 18. Expanded genomic structure of exons 17 to 19. Exon 18 N contains an in frame stop codon. Splice variants generated by alternative splicing of Exon 18 are indicated with the PCR product length expected by using primers located in Exons 17 and 19 (see Methods). ( B – E ) End-point PCR electrophoresis results for SCN8A exon 18 variants expressed in non-cancerous cervix, cervical intraepithelial neoplasia, invasive cervical cancer, and cervical cancer cell lines, respectively. HEK-Nav1.6 cells was used as positive control for the adult splice form of SCN8A (18A; far-right line). A 100-bp molecular weight marker was used as reference (far-left line). The SCN8A variants generated for alternative splicing of exon 18: 18A, 18N and Δ18, were identified in the indicated group of samples. Identity of the SCN8A splice forms was confirmed by automated sequencing. The SCN8A splice forms were relatively more abundant in human cervical cancer samples, more clearly for the Δ18 variant; whereas the adult (18A) variant was practically absent in CeCa cell lines. From the two samples (266 and 275) that were present in all western blots experiments, only mRNA from sample 266 was available for performing these PCR analysis.
    Figure Legend Snippet: Alternative splicing of SCN8A exon 18 in the neoplasia-carcinoma sequence of human cervical tissue. ( A ) Alternative splicing of SCN8A Exon 18. Expanded genomic structure of exons 17 to 19. Exon 18 N contains an in frame stop codon. Splice variants generated by alternative splicing of Exon 18 are indicated with the PCR product length expected by using primers located in Exons 17 and 19 (see Methods). ( B – E ) End-point PCR electrophoresis results for SCN8A exon 18 variants expressed in non-cancerous cervix, cervical intraepithelial neoplasia, invasive cervical cancer, and cervical cancer cell lines, respectively. HEK-Nav1.6 cells was used as positive control for the adult splice form of SCN8A (18A; far-right line). A 100-bp molecular weight marker was used as reference (far-left line). The SCN8A variants generated for alternative splicing of exon 18: 18A, 18N and Δ18, were identified in the indicated group of samples. Identity of the SCN8A splice forms was confirmed by automated sequencing. The SCN8A splice forms were relatively more abundant in human cervical cancer samples, more clearly for the Δ18 variant; whereas the adult (18A) variant was practically absent in CeCa cell lines. From the two samples (266 and 275) that were present in all western blots experiments, only mRNA from sample 266 was available for performing these PCR analysis.

    Techniques Used: Sequencing, Generated, Polymerase Chain Reaction, Electrophoresis, Positive Control, Molecular Weight, Marker, Variant Assay, Western Blot

    3) Product Images from "Dominance of HIV-1 Subtype CRF01_AE in Sexually Acquired Cases Leads to a New Epidemic in Yunnan Province of China"

    Article Title: Dominance of HIV-1 Subtype CRF01_AE in Sexually Acquired Cases Leads to a New Epidemic in Yunnan Province of China

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0030443

    Phylogenetic Neighbor-Joining Tree for HIV-1 p17 Sequences Obtained from All 16 Prefectures of Yunnan Individual sequences are color coded, with the colors corresponding to those of original geographic sites on the map of Yunnan ( Figure 1 ). The horizontal branch was drawn in accordance with their relative genetic distances. The vertical lines are present purely for clarity of the tree presentation. The bootstrap values of 1,000 replicates are labeled on the major branches. The reference sequences for classifying HIV-1 genotypes were included and were originally obtained from the NIH/NIAID–funded HIV Databases.
    Figure Legend Snippet: Phylogenetic Neighbor-Joining Tree for HIV-1 p17 Sequences Obtained from All 16 Prefectures of Yunnan Individual sequences are color coded, with the colors corresponding to those of original geographic sites on the map of Yunnan ( Figure 1 ). The horizontal branch was drawn in accordance with their relative genetic distances. The vertical lines are present purely for clarity of the tree presentation. The bootstrap values of 1,000 replicates are labeled on the major branches. The reference sequences for classifying HIV-1 genotypes were included and were originally obtained from the NIH/NIAID–funded HIV Databases.

    Techniques Used: Labeling

    4) Product Images from "Neodiversification of homeologous CLAVATA1-like receptor kinase genes in soybean leads to distinct developmental outcomes"

    Article Title: Neodiversification of homeologous CLAVATA1-like receptor kinase genes in soybean leads to distinct developmental outcomes

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-08252-y

    Transcript levels of GmCLV1A and GmTrCLV1A in various tissues of 14 day-old, uninoculated soybean plants. Values were measured using qRT-PCR; n = 3 biological replicates per tissue; error bars indicate SE. TR1 = first 2 cm from taproot tip; TR2 = second 2 cm from taproot tip; LR1 = first 2 cm from lateral root tip; LR2 = second 2 cm from lateral root tip; UF = unifoliate leaf; TF = trifoliate leaf; Vein = vein of trifoliate leaf; Hypo = hypocotyls; Stem = stem above hypocotyl; STip = shoot tip. Note the 10-fold difference in scale.
    Figure Legend Snippet: Transcript levels of GmCLV1A and GmTrCLV1A in various tissues of 14 day-old, uninoculated soybean plants. Values were measured using qRT-PCR; n = 3 biological replicates per tissue; error bars indicate SE. TR1 = first 2 cm from taproot tip; TR2 = second 2 cm from taproot tip; LR1 = first 2 cm from lateral root tip; LR2 = second 2 cm from lateral root tip; UF = unifoliate leaf; TF = trifoliate leaf; Vein = vein of trifoliate leaf; Hypo = hypocotyls; Stem = stem above hypocotyl; STip = shoot tip. Note the 10-fold difference in scale.

    Techniques Used: Quantitative RT-PCR

    Phenotypes of pod, stem (as demonstrated by cotyledonary node branching) and nodulated root systems of the soybean wild type Forrest, and its TILLING-derived mutants , Gmclv1a ( S562L) and Gmnark ( W677* ).
    Figure Legend Snippet: Phenotypes of pod, stem (as demonstrated by cotyledonary node branching) and nodulated root systems of the soybean wild type Forrest, and its TILLING-derived mutants , Gmclv1a ( S562L) and Gmnark ( W677* ).

    Techniques Used: Derivative Assay

    Phenotypes of reciprocally grafted (scion/rootstock) plants between wild-type soybean cv. Forrest and its mutants Gmclv1a ( S562L ) Gmnark ( W677* ). Plants were grafted 12 days after sowing. Data were collected 45 days later. ( A) Nodule number per plant; ( B) lateral root number per plant (in the 5–15 cm region below the crown); ( C) average nodule weight; and ( D) nodulation index ( i.e ., % of root nodulated). Different letters above the bar represent statistically significant differences (Duncan test; P ≤ 0.05). Error bars indicate SE.
    Figure Legend Snippet: Phenotypes of reciprocally grafted (scion/rootstock) plants between wild-type soybean cv. Forrest and its mutants Gmclv1a ( S562L ) Gmnark ( W677* ). Plants were grafted 12 days after sowing. Data were collected 45 days later. ( A) Nodule number per plant; ( B) lateral root number per plant (in the 5–15 cm region below the crown); ( C) average nodule weight; and ( D) nodulation index ( i.e ., % of root nodulated). Different letters above the bar represent statistically significant differences (Duncan test; P ≤ 0.05). Error bars indicate SE.

    Techniques Used:

    Temperature influence on phenotypes of wild-type soybean cv. Forrest, and its mutants Gmclv1a ( S562L ), Gmnark ( W677* ) and the Gmclv1a Gmnark double mutant (DM). Plants were grown at 28/25 °C or 20/17 °C. ( A) Plant height. ( B) Node number. ( C) Leaf number at node 3. ( D) Percentage of plants having at least one vein-bladed leaf; Vein-bladed phenotype were scored 4 weeks after flowering. ( E) Pod number (including both developing and mature pods). ( F) Nodule number per plant. ( G) Shoot dry weight. ( H) Root dry weight. Plant height, node number and leaf number at node 3 were measured 4 weeks after sowing; n = 9–13. Nodule number, shoot and root dry weight were measured 3 weeks after sowing; n = 6. Error bars indicate SE. Nd = ‘not detected’. Different letters above the bar represent statistically significant differences (Duncan test; P ≤ 0.05).
    Figure Legend Snippet: Temperature influence on phenotypes of wild-type soybean cv. Forrest, and its mutants Gmclv1a ( S562L ), Gmnark ( W677* ) and the Gmclv1a Gmnark double mutant (DM). Plants were grown at 28/25 °C or 20/17 °C. ( A) Plant height. ( B) Node number. ( C) Leaf number at node 3. ( D) Percentage of plants having at least one vein-bladed leaf; Vein-bladed phenotype were scored 4 weeks after flowering. ( E) Pod number (including both developing and mature pods). ( F) Nodule number per plant. ( G) Shoot dry weight. ( H) Root dry weight. Plant height, node number and leaf number at node 3 were measured 4 weeks after sowing; n = 9–13. Nodule number, shoot and root dry weight were measured 3 weeks after sowing; n = 6. Error bars indicate SE. Nd = ‘not detected’. Different letters above the bar represent statistically significant differences (Duncan test; P ≤ 0.05).

    Techniques Used: Mutagenesis

    Structural aspects of GmCLV1A and GmTrCLV1A. ( A) Predicted model of the extracellular LRR domain of GmCLV1A , including the site of the S562L mis-sense mutation. The amino acid highlighted in red represent the serine of the predicted glycosylation site that is mutated to a leucine in S562L ( B) Predicted protein domains. SP = signal peptide; LRRNT_2 = Leucine-rich repeat N-terminal; TM = Transmembrane domain. ( C) Protein alignment of the mutated region of S562L compared with that of AtCLV1, GmCLV1A , GmTrCLV1A, GmNARK, MtSUNN, and MtRLP1. The red box highlights the predicted glycosylation site.
    Figure Legend Snippet: Structural aspects of GmCLV1A and GmTrCLV1A. ( A) Predicted model of the extracellular LRR domain of GmCLV1A , including the site of the S562L mis-sense mutation. The amino acid highlighted in red represent the serine of the predicted glycosylation site that is mutated to a leucine in S562L ( B) Predicted protein domains. SP = signal peptide; LRRNT_2 = Leucine-rich repeat N-terminal; TM = Transmembrane domain. ( C) Protein alignment of the mutated region of S562L compared with that of AtCLV1, GmCLV1A , GmTrCLV1A, GmNARK, MtSUNN, and MtRLP1. The red box highlights the predicted glycosylation site.

    Techniques Used: Mutagenesis

    Macro- and microscopic phenotypes of the soybean wild type Forrest, and its mutant S562L . ( A) Stem thickness of 5 month-old plants (plants were intentionally defoliated to enhance visibility of stem architecture); ( B) First trifoliate node showing fasciation and excessive flowering in the mutant; ( C) Vein-bladed leaf structures on the underside of Gmclv1a mutant leaves. ( D) Young pod morphology (dashed line indicates the position of the cross-section seen in ( F ). ( E) Stem section at node 4 of Forrest and S562L mutant plants (4 month-old). ( F) Young pod cross-sections. Note the bifurcated, deformed pod of the S562L mutant. VB = Vascular bundle; Ep = Epidermis; IS = Inner sclerenchyma.
    Figure Legend Snippet: Macro- and microscopic phenotypes of the soybean wild type Forrest, and its mutant S562L . ( A) Stem thickness of 5 month-old plants (plants were intentionally defoliated to enhance visibility of stem architecture); ( B) First trifoliate node showing fasciation and excessive flowering in the mutant; ( C) Vein-bladed leaf structures on the underside of Gmclv1a mutant leaves. ( D) Young pod morphology (dashed line indicates the position of the cross-section seen in ( F ). ( E) Stem section at node 4 of Forrest and S562L mutant plants (4 month-old). ( F) Young pod cross-sections. Note the bifurcated, deformed pod of the S562L mutant. VB = Vascular bundle; Ep = Epidermis; IS = Inner sclerenchyma.

    Techniques Used: Mutagenesis

    Structure and genomic environments of CLAVATA1 and AON-related genes. ( A) Intron and exon positions and sizes of AtCLV1 , GmCLV1A, GmNARK, MtSUNN, LjHAR1, PsSYM29 and PvNARK . ( B ) TILLed regions of GmNARK and GmCLV1A . ( C ) Genomic environment of AtCLV1A , GmCLV1A , GmNARK , LiHAR1 , MtSUNN and PvNARK ; approximately 100 kb is shown. The same number and colour indicates similar genes. The CLV1 and its orthologs in legumes are in grey. The number ‘1’ represents a truncated gene. ( D ) Positioning and size of GmCLV1A with GmTrCLV1A, PvNARK with PvTrNARK and MtSUNN with MtRLP1 .
    Figure Legend Snippet: Structure and genomic environments of CLAVATA1 and AON-related genes. ( A) Intron and exon positions and sizes of AtCLV1 , GmCLV1A, GmNARK, MtSUNN, LjHAR1, PsSYM29 and PvNARK . ( B ) TILLed regions of GmNARK and GmCLV1A . ( C ) Genomic environment of AtCLV1A , GmCLV1A , GmNARK , LiHAR1 , MtSUNN and PvNARK ; approximately 100 kb is shown. The same number and colour indicates similar genes. The CLV1 and its orthologs in legumes are in grey. The number ‘1’ represents a truncated gene. ( D ) Positioning and size of GmCLV1A with GmTrCLV1A, PvNARK with PvTrNARK and MtSUNN with MtRLP1 .

    Techniques Used:

    Branching phenotype of 4 week-old soybean cv. Forrest, its mutant Gmclv1a ( S562L ), and F 2 segregants from a cross between them. CC = wild-type segregants; cc = Gmclv1a segregants. Forrest n = 10, S562L n = 9, CC n = 11 and cc n = 14. Error bars indicate SE. Different letters above bars represent statistically significant differences (Student’s t test; P ≤ 0.05).
    Figure Legend Snippet: Branching phenotype of 4 week-old soybean cv. Forrest, its mutant Gmclv1a ( S562L ), and F 2 segregants from a cross between them. CC = wild-type segregants; cc = Gmclv1a segregants. Forrest n = 10, S562L n = 9, CC n = 11 and cc n = 14. Error bars indicate SE. Different letters above bars represent statistically significant differences (Student’s t test; P ≤ 0.05).

    Techniques Used: Mutagenesis

    5) Product Images from "Characterization of Glycosomal RING Finger Proteins of Trypanosomatids"

    Article Title: Characterization of Glycosomal RING Finger Proteins of Trypanosomatids

    Journal:

    doi: 10.1016/j.exppara.2006.11.004

    GFP-PEX10 and GFP-PEX12 membrane topology
    Figure Legend Snippet: GFP-PEX10 and GFP-PEX12 membrane topology

    Techniques Used:

    Localization of PEX10 and PEX12 GFP fusion proteins expressed in T. brucei
    Figure Legend Snippet: Localization of PEX10 and PEX12 GFP fusion proteins expressed in T. brucei

    Techniques Used:

    6) Product Images from "Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A) *"

    Article Title: Ectodomain Shedding of Lymphatic Vessel Endothelial Hyaluronan Receptor 1 (LYVE-1) Is Induced by Vascular Endothelial Growth Factor A (VEGF-A) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.683201

    Targeted overexpression of VEGF-A in mouse skin is associated with extracellular localization of LYVE-1. A and B , wild-type littermates show normal ear skin ( A ), whereas targeted overexpression of VEGF-A leads to erythematous plaques resembling psoriasis ( B ). C–F , colloidal carbon injection ( asterisk ) into the ears revealed that VEGF-A overexpression impaired fluid transport by cutaneous lymphatic vessels ( F , arrowheads ). Respective images indicate 5 ( C and D ) or 30 min ( E and F ) after injection. Scale bar , 1 mm. G–J , H E and double immunofluorescence staining for blood (PV-1; green ) and lymphatic vessels (LYVE-1; red ) shows prominent expansion of lymphatic vessels without continuous vessel wall in VEGF-A transgenic mice ( J , arrowheads ). Nuclei were stained by DAPI ( blue ). Scale bar , 100 μm. K–N , electron microscopic analyses showed that VEGF-A overexpression leads to fenestration of initial lymphatics that sometimes lack overlap of lymphatic endothelial cells ( N , arrows ). Scale bar , 100 μm. O and P , three-dimensional confocal microscopic images of LYVE-1 ( red ) and claudin-5 ( green ) show prominent extracellular localization of LYVE-1 in VEGF-A transgenic mice. TG , transgenic.
    Figure Legend Snippet: Targeted overexpression of VEGF-A in mouse skin is associated with extracellular localization of LYVE-1. A and B , wild-type littermates show normal ear skin ( A ), whereas targeted overexpression of VEGF-A leads to erythematous plaques resembling psoriasis ( B ). C–F , colloidal carbon injection ( asterisk ) into the ears revealed that VEGF-A overexpression impaired fluid transport by cutaneous lymphatic vessels ( F , arrowheads ). Respective images indicate 5 ( C and D ) or 30 min ( E and F ) after injection. Scale bar , 1 mm. G–J , H E and double immunofluorescence staining for blood (PV-1; green ) and lymphatic vessels (LYVE-1; red ) shows prominent expansion of lymphatic vessels without continuous vessel wall in VEGF-A transgenic mice ( J , arrowheads ). Nuclei were stained by DAPI ( blue ). Scale bar , 100 μm. K–N , electron microscopic analyses showed that VEGF-A overexpression leads to fenestration of initial lymphatics that sometimes lack overlap of lymphatic endothelial cells ( N , arrows ). Scale bar , 100 μm. O and P , three-dimensional confocal microscopic images of LYVE-1 ( red ) and claudin-5 ( green ) show prominent extracellular localization of LYVE-1 in VEGF-A transgenic mice. TG , transgenic.

    Techniques Used: Over Expression, Injection, Double Immunofluorescence Staining, Transgenic Assay, Mouse Assay, Staining

    Expression patterns of LYVE-1 in psoriasis skin. A , ELISA analysis revealed reactivity against human ( hLYVE-1 ) and mouse LYVE-1 ( mLYVE-1 ) CTF but not against human CD44 ( hCD44 ) CTF. B and C , double immunofluorescence analysis for LYVE-1 ( red ) and PV-1 ( green ) in normal ( A ) and psoriatic human skin ( B ). Nuclei were stained by DAPI ( blue ). Scale bar , 100 μm. D and E , immunofluorescence staining for LYVE-1 ( grayscale ) in normal ( D ) and psoriatic human skin ( E ). Scale bar , 100 μm. F–K , double immunofluorescence staining for the extracellular domain ( ECD ; red ) and CTF ( green ) of LYVE-1 in normal and psoriasis skin. Scale bar , 30 μm. L–Q , double immunofluorescence staining for the extracellular domain ( red ) and CTF ( green ) of LYVE-1 in wild-type and VEGF-A transgenic ( TG ) mouse skin. Scale bar , 10 μm.
    Figure Legend Snippet: Expression patterns of LYVE-1 in psoriasis skin. A , ELISA analysis revealed reactivity against human ( hLYVE-1 ) and mouse LYVE-1 ( mLYVE-1 ) CTF but not against human CD44 ( hCD44 ) CTF. B and C , double immunofluorescence analysis for LYVE-1 ( red ) and PV-1 ( green ) in normal ( A ) and psoriatic human skin ( B ). Nuclei were stained by DAPI ( blue ). Scale bar , 100 μm. D and E , immunofluorescence staining for LYVE-1 ( grayscale ) in normal ( D ) and psoriatic human skin ( E ). Scale bar , 100 μm. F–K , double immunofluorescence staining for the extracellular domain ( ECD ; red ) and CTF ( green ) of LYVE-1 in normal and psoriasis skin. Scale bar , 30 μm. L–Q , double immunofluorescence staining for the extracellular domain ( red ) and CTF ( green ) of LYVE-1 in wild-type and VEGF-A transgenic ( TG ) mouse skin. Scale bar , 10 μm.

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Double Immunofluorescence Staining, Transgenic Assay

    7) Product Images from "The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton ▿"

    Article Title: The N Terminus of Phosphodiesterase TbrPDEB1 of Trypanosoma brucei Contains the Signal for Integration into the Flagellar Skeleton ▿

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00112-10

    Schematic representation of constructs. FL, full-length PDEB1 or PDEB2; open circle, C-terminal c-Myc or HA tag; B1(1–212)::GFP, B1(1–114)::GFP, and B1(1–70)::GFP, GFP fusions carrying the N-terminal 212, 114, and 70 amino acids
    Figure Legend Snippet: Schematic representation of constructs. FL, full-length PDEB1 or PDEB2; open circle, C-terminal c-Myc or HA tag; B1(1–212)::GFP, B1(1–114)::GFP, and B1(1–70)::GFP, GFP fusions carrying the N-terminal 212, 114, and 70 amino acids

    Techniques Used: Construct

    The N terminus of PDEB1, but not that of PDEB2, confers integration into the flagellar skeleton. (A) Amino acids 1 to 212 of PDEB1 lead to stable integration of the GFP reporter into the flagellar skeleton; (B) amino acids 1 to 212 of PDEB2 do not; (C)
    Figure Legend Snippet: The N terminus of PDEB1, but not that of PDEB2, confers integration into the flagellar skeleton. (A) Amino acids 1 to 212 of PDEB1 lead to stable integration of the GFP reporter into the flagellar skeleton; (B) amino acids 1 to 212 of PDEB2 do not; (C)

    Techniques Used:

    8) Product Images from "A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides"

    Article Title: A Family of Helminth Molecules that Modulate Innate Cell Responses via Molecular Mimicry of Host Antimicrobial Peptides

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002042

    Phylogenetic relationships of the HDMs. (A) A bootstrapped (1000 trials) neighbour-joining phylogenetic tree showing the evolutionary relationship of HDM cDNA sequences from medically-important trematode pathogens. Numbers represent bootstrap values (given as percentages) for a particular node, and values greater than 65% are shown. The tree is rooted to human CAP18 (accession number NM_004345). Three major clades are shown corresponding to the Sm16-like molecules, the schistosome HDMs and HDMs from Fasciola and the Asian flukes. (B) Primary sequence alignment of selected members of the HDM clades. Conserved residues that contribute to the hydrophobic face of the amphipathic helix are shaded in grey. (C) Top panel. RT-PCR analysis of FhHDM-1 expression in F. hepatica newly excysted juveniles (NEJ), 21-day immature flukes (21d) and adult worms (Adult). Amplification of constitutively expressed F. hepatica β-actin was performed as a positive control. Samples were separated by agarose gel electrophoresis and stained with ethidium bromide. Bottom panel. Immunogenicity of FhHDM-1 in F. hepatica -infected sheep. Pre-infection sera (Pre) and samples taken 4, 8, 12 and 16 weeks post-infection were analysed by ELISA and Western blot using an anti-FhHDM-1 antibody. Specific antibody responses were detected at week 4 with immunoblot staining stronger at weeks 8 and 12 after infection.
    Figure Legend Snippet: Phylogenetic relationships of the HDMs. (A) A bootstrapped (1000 trials) neighbour-joining phylogenetic tree showing the evolutionary relationship of HDM cDNA sequences from medically-important trematode pathogens. Numbers represent bootstrap values (given as percentages) for a particular node, and values greater than 65% are shown. The tree is rooted to human CAP18 (accession number NM_004345). Three major clades are shown corresponding to the Sm16-like molecules, the schistosome HDMs and HDMs from Fasciola and the Asian flukes. (B) Primary sequence alignment of selected members of the HDM clades. Conserved residues that contribute to the hydrophobic face of the amphipathic helix are shaded in grey. (C) Top panel. RT-PCR analysis of FhHDM-1 expression in F. hepatica newly excysted juveniles (NEJ), 21-day immature flukes (21d) and adult worms (Adult). Amplification of constitutively expressed F. hepatica β-actin was performed as a positive control. Samples were separated by agarose gel electrophoresis and stained with ethidium bromide. Bottom panel. Immunogenicity of FhHDM-1 in F. hepatica -infected sheep. Pre-infection sera (Pre) and samples taken 4, 8, 12 and 16 weeks post-infection were analysed by ELISA and Western blot using an anti-FhHDM-1 antibody. Specific antibody responses were detected at week 4 with immunoblot staining stronger at weeks 8 and 12 after infection.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Positive Control, Agarose Gel Electrophoresis, Staining, Infection, Enzyme-linked Immunosorbent Assay, Western Blot

    Identification and characterisation of native FhHDM-1. (A) Secretory proteins collected from adult F. hepatica following in vitro culture were separated by gel filtration and the resulting high molecular mass ( > 200 kDa) peak (peak I; PI) was separated further using reverse phase HLPC (RP-HPLC). Fractions collected following gel filtration and RP-HPLC were run on reducing 4–12% Bis-Tris gels (B) and showed that a prominent ∼ 6 kDa protein present in total adult secretory proteins (S) was enriched in PI and purified to homogeneity ( > 95%) following RP-HPLC (E). (C) Western blot of adult fluke secretions probed with an anti-FhHDM-1 antibody. P, pre-immune sera; T, test bleed. (D) N-terminal sequencing and LC-MS/MS analysis of the native ∼ 6 kDa protein generated peptide sequence information that allowed cloning of the cDNA, termed FhHDM-1. The primary amino acid sequence of FhHDM-1 derived from conceptual translation of the cDNA is shown. The predicted N-terminal signal peptide is shown in italics and the actual N-terminal of the native protein is shown by an arrow. The SEESREKLRE sequence generated by N-terminal sequencing is boxed in grey and a peptide ( m/z 642.93; ITEVITILLNR) matched by LC-MS/MS following tryptic digest of the native protein is underlined. Secondary structure predictions using using PSIPRED [26] , shown below the primary sequence, suggest the molecule is predominantly α-helical.
    Figure Legend Snippet: Identification and characterisation of native FhHDM-1. (A) Secretory proteins collected from adult F. hepatica following in vitro culture were separated by gel filtration and the resulting high molecular mass ( > 200 kDa) peak (peak I; PI) was separated further using reverse phase HLPC (RP-HPLC). Fractions collected following gel filtration and RP-HPLC were run on reducing 4–12% Bis-Tris gels (B) and showed that a prominent ∼ 6 kDa protein present in total adult secretory proteins (S) was enriched in PI and purified to homogeneity ( > 95%) following RP-HPLC (E). (C) Western blot of adult fluke secretions probed with an anti-FhHDM-1 antibody. P, pre-immune sera; T, test bleed. (D) N-terminal sequencing and LC-MS/MS analysis of the native ∼ 6 kDa protein generated peptide sequence information that allowed cloning of the cDNA, termed FhHDM-1. The primary amino acid sequence of FhHDM-1 derived from conceptual translation of the cDNA is shown. The predicted N-terminal signal peptide is shown in italics and the actual N-terminal of the native protein is shown by an arrow. The SEESREKLRE sequence generated by N-terminal sequencing is boxed in grey and a peptide ( m/z 642.93; ITEVITILLNR) matched by LC-MS/MS following tryptic digest of the native protein is underlined. Secondary structure predictions using using PSIPRED [26] , shown below the primary sequence, suggest the molecule is predominantly α-helical.

    Techniques Used: In Vitro, Filtration, High Performance Liquid Chromatography, Purification, Western Blot, Sequencing, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Generated, Clone Assay, Derivative Assay

    FhCL1 processes FhHDM-1 at low pH. (A) Total secretory proteins from adult F. hepatica were concentrated from culture supernatants using a 3 kDa cut-off filter. 10 µg of the flow-through (FT) was analysed on a 4–12% Bi-Tris gel and stained with Flamingo fluorescent protein stain. The FT comprised a single prominent band (∼ 3.5 kDa) that was identified by LC-MS/MS as an FhHDM-1 fragment (high-scoring peptide ITEVITILLNR; m/z 642.93, underlined in D). N-terminal sequencing of this band was unsuccessful. (B) To investigate whether F. hepatica cathepsin L1 (FhCL1) can process FhHDM-1, 50 µg recombinant FhHDM-1 was incubated with 1 µg recombinant FhCL1 [35] in either 0.1 M sodium acetate (pH 4.5) or 0.1 M sodium phosphate (pH 7.3) each containing 1 mM EDTA and 1 mM DTT. Reactions were performed ± FhCL1 for 3 h at 37°C and stopped by the addition of E-64 (10 µM). Samples were analysed on 4–12% Bis-Tris gels and blots were probed with anti-His or anti-FhHDM-1 antibodies. (C) The pH 4.5 reaction in the presence of FhCL1 shown in (B) was analysed by MALDI-TOF MS. The major masses detected correspond to the full length recombinant FhHDM-1 ± the C-terminal His-tag ( m/z 9272.88 and 8450.53 respectively) and two fragments (both m/z 4232.44) created by a single cleavage after Arg 56 (native peptide numbering). (D) The putative FhHDM-1 cleavage sites are arrowed. Based on this, the synthetic peptide FhHDM-1 p2 was designed (shown as a cartoon above the primary sequence of recombinant FhHDM-1). Whilst trypsinising recombinant FhHDM-1 considerably reduced its interaction with LPS, boiling had no effect.
    Figure Legend Snippet: FhCL1 processes FhHDM-1 at low pH. (A) Total secretory proteins from adult F. hepatica were concentrated from culture supernatants using a 3 kDa cut-off filter. 10 µg of the flow-through (FT) was analysed on a 4–12% Bi-Tris gel and stained with Flamingo fluorescent protein stain. The FT comprised a single prominent band (∼ 3.5 kDa) that was identified by LC-MS/MS as an FhHDM-1 fragment (high-scoring peptide ITEVITILLNR; m/z 642.93, underlined in D). N-terminal sequencing of this band was unsuccessful. (B) To investigate whether F. hepatica cathepsin L1 (FhCL1) can process FhHDM-1, 50 µg recombinant FhHDM-1 was incubated with 1 µg recombinant FhCL1 [35] in either 0.1 M sodium acetate (pH 4.5) or 0.1 M sodium phosphate (pH 7.3) each containing 1 mM EDTA and 1 mM DTT. Reactions were performed ± FhCL1 for 3 h at 37°C and stopped by the addition of E-64 (10 µM). Samples were analysed on 4–12% Bis-Tris gels and blots were probed with anti-His or anti-FhHDM-1 antibodies. (C) The pH 4.5 reaction in the presence of FhCL1 shown in (B) was analysed by MALDI-TOF MS. The major masses detected correspond to the full length recombinant FhHDM-1 ± the C-terminal His-tag ( m/z 9272.88 and 8450.53 respectively) and two fragments (both m/z 4232.44) created by a single cleavage after Arg 56 (native peptide numbering). (D) The putative FhHDM-1 cleavage sites are arrowed. Based on this, the synthetic peptide FhHDM-1 p2 was designed (shown as a cartoon above the primary sequence of recombinant FhHDM-1). Whilst trypsinising recombinant FhHDM-1 considerably reduced its interaction with LPS, boiling had no effect.

    Techniques Used: Flow Cytometry, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Sequencing, Recombinant, Incubation

    LPS neutralisation by FhHDM-1. (A) Alignment of full-length FhHDM-1 with peptide 1 (FhHDM-1 p1) and peptide 2 (FhHDM-1 p2). The conserved C-terminal amphipathic helix is shaded in grey. (B) The ability of native and recombinant FhHDM-1 to bind LPS was investigated by incubating the proteins (2 µg/well) in an LPS-coated (100 ng/well) microtitre plate. Bound proteins were detected by ELISA using rabbit anti-FhHDM-1 as a primary antibody. BSA was used as a baseline control. Whilst trypsinising the recombinant FhHDM-1 significantly reduced the LPS interaction, boiling had no effect. (C) The ability of recombinant FhHDM-1 (Δ), FhHDM-1 p1 (•) or FhHDM-1 p2 (▪) to bind to LPS was investigated by incubating a range of concentrations of proteins (0.02–2 µg/well) in this assay. (D) FhHDM-1 or derived peptides (0.1 µg) were incubated in the presence of LPS (0.05-5 µg/well) and bound peptides measured as described above. Binding of peptides to the LPS-immobilised plates was expressed as a percentage of that measured for 2 µg (for panel C) or 0.1 µg (for panel D) of FhHDM-1. Data are the means ± SD from three separate experiments. (E) FhHDM-1 and FhHDM-1 p2 but not FhHDM-1 p1 reduced the interaction between LPS and LBP as effectively as LL-37. LPS-coated microtitre plates were incubated with 5 µg/well of F. hepatica ES, LL-37, FhHDM-1 or derived peptides for 1 h prior to the addition of 10% human sera in PBS. Interaction of LBP with LPS was measured by ELISA using an anti-LBP primary antibody and expressed as a percentage of that detected for 10% sera in the absence of added peptides. Data are the mean ± SD of three separate experiments. Statistical significance was calculated using the student t-test and represent a comparison to the binding of 10% sera to immobilised LPS. (F) Binding of FITC-conjugated LPS to RAW264.7 cells was inhibited by LL-37, FhHDM-1 and peptides. RAW264.7 cells (5×10 5 cells/ml) were incubated with 100 ng/ml of FITC-conjugated LPS in the presence of FhHDM-1, FhHDM-1 p1, FhHDM-1 p2 and LL-37 (5 µg/ml) in RPMI 1640 containing 10% FBS for 20 min at 4°C. The binding of FITC-LPS was analysed by flow cytometry. Values represent percentage inhibition of FITC-LPS binding compared to cells in the absence of peptides. Data are the mean fluorescence ± SD of three independent experiments.
    Figure Legend Snippet: LPS neutralisation by FhHDM-1. (A) Alignment of full-length FhHDM-1 with peptide 1 (FhHDM-1 p1) and peptide 2 (FhHDM-1 p2). The conserved C-terminal amphipathic helix is shaded in grey. (B) The ability of native and recombinant FhHDM-1 to bind LPS was investigated by incubating the proteins (2 µg/well) in an LPS-coated (100 ng/well) microtitre plate. Bound proteins were detected by ELISA using rabbit anti-FhHDM-1 as a primary antibody. BSA was used as a baseline control. Whilst trypsinising the recombinant FhHDM-1 significantly reduced the LPS interaction, boiling had no effect. (C) The ability of recombinant FhHDM-1 (Δ), FhHDM-1 p1 (•) or FhHDM-1 p2 (▪) to bind to LPS was investigated by incubating a range of concentrations of proteins (0.02–2 µg/well) in this assay. (D) FhHDM-1 or derived peptides (0.1 µg) were incubated in the presence of LPS (0.05-5 µg/well) and bound peptides measured as described above. Binding of peptides to the LPS-immobilised plates was expressed as a percentage of that measured for 2 µg (for panel C) or 0.1 µg (for panel D) of FhHDM-1. Data are the means ± SD from three separate experiments. (E) FhHDM-1 and FhHDM-1 p2 but not FhHDM-1 p1 reduced the interaction between LPS and LBP as effectively as LL-37. LPS-coated microtitre plates were incubated with 5 µg/well of F. hepatica ES, LL-37, FhHDM-1 or derived peptides for 1 h prior to the addition of 10% human sera in PBS. Interaction of LBP with LPS was measured by ELISA using an anti-LBP primary antibody and expressed as a percentage of that detected for 10% sera in the absence of added peptides. Data are the mean ± SD of three separate experiments. Statistical significance was calculated using the student t-test and represent a comparison to the binding of 10% sera to immobilised LPS. (F) Binding of FITC-conjugated LPS to RAW264.7 cells was inhibited by LL-37, FhHDM-1 and peptides. RAW264.7 cells (5×10 5 cells/ml) were incubated with 100 ng/ml of FITC-conjugated LPS in the presence of FhHDM-1, FhHDM-1 p1, FhHDM-1 p2 and LL-37 (5 µg/ml) in RPMI 1640 containing 10% FBS for 20 min at 4°C. The binding of FITC-LPS was analysed by flow cytometry. Values represent percentage inhibition of FITC-LPS binding compared to cells in the absence of peptides. Data are the mean fluorescence ± SD of three independent experiments.

    Techniques Used: Recombinant, Enzyme-linked Immunosorbent Assay, Derivative Assay, Incubation, Binding Assay, Flow Cytometry, Cytometry, Inhibition, Fluorescence

    FhHDM-1 is structurally homologous with LL-37. (A) Primary sequence alignment of FhHDM-1 with the human LL-37 precursor, hCAP18. The LL-37 processing site is arrowed. (B) Helical wheel analysis shows that the conserved C-terminal hydrophobic regions boxed in (A) form amphipathic helices in both molecules.
    Figure Legend Snippet: FhHDM-1 is structurally homologous with LL-37. (A) Primary sequence alignment of FhHDM-1 with the human LL-37 precursor, hCAP18. The LL-37 processing site is arrowed. (B) Helical wheel analysis shows that the conserved C-terminal hydrophobic regions boxed in (A) form amphipathic helices in both molecules.

    Techniques Used: Sequencing

    Sedimentation velocity analysis of recombinant FhHDM-1. (A) Continuous size-distribution analysis, c(s) , plotted as a function of sedimentation coefficient for recombinant FhHDM-1 at pH 4.5 (solid line) and pH 7.3 (dashed line). Continuous size-distribution analysis was performed using the program SEDFIT [29] – [31] employing 100 sedimentation coefficients ranging from 0.1 S to 6.0 S and at a confidence level (F-ratio) = 0.95. (B) Continuous mass, c(M) , distribution plotted as a function of molecular mass (kDa) for recombinant FhHDM-1 at pH 4.5 (solid line) and pH 7.3 (dashed line). Continuous mass-distribution analysis was performed using SEDFIT with 100 masses ranging from 1.0 kDa to 80 kDa and at a confidence level (F-ratio) = 0.95.
    Figure Legend Snippet: Sedimentation velocity analysis of recombinant FhHDM-1. (A) Continuous size-distribution analysis, c(s) , plotted as a function of sedimentation coefficient for recombinant FhHDM-1 at pH 4.5 (solid line) and pH 7.3 (dashed line). Continuous size-distribution analysis was performed using the program SEDFIT [29] – [31] employing 100 sedimentation coefficients ranging from 0.1 S to 6.0 S and at a confidence level (F-ratio) = 0.95. (B) Continuous mass, c(M) , distribution plotted as a function of molecular mass (kDa) for recombinant FhHDM-1 at pH 4.5 (solid line) and pH 7.3 (dashed line). Continuous mass-distribution analysis was performed using SEDFIT with 100 masses ranging from 1.0 kDa to 80 kDa and at a confidence level (F-ratio) = 0.95.

    Techniques Used: Sedimentation, Recombinant

    FhHDM-1 protects mice from LPS-induced inflammation. (A) BALB/c mice were injected intra-peritoneally with 1 µg of LPS alone or combined with 1 µg of FhHDM-1, FhHDM-1 p2 or LL-37. Two hours later, sera was collected and serum levels of TNF and (B) IL-1β measured by ELISA. (C) Peritoneal macrophages were isolated, cultured unstimulated in media overnight and then levels of TNF and (D) IL-1β in the culture measured by ELISA. Data are the mean ± SD of six mice in each group. Statistical significance represents a comparison to the levels of cytokines secreted by mice given LPS only.
    Figure Legend Snippet: FhHDM-1 protects mice from LPS-induced inflammation. (A) BALB/c mice were injected intra-peritoneally with 1 µg of LPS alone or combined with 1 µg of FhHDM-1, FhHDM-1 p2 or LL-37. Two hours later, sera was collected and serum levels of TNF and (B) IL-1β measured by ELISA. (C) Peritoneal macrophages were isolated, cultured unstimulated in media overnight and then levels of TNF and (D) IL-1β in the culture measured by ELISA. Data are the mean ± SD of six mice in each group. Statistical significance represents a comparison to the levels of cytokines secreted by mice given LPS only.

    Techniques Used: Mouse Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture

    Expression and CD spectroscopy of recombinant FhHDM-1. (A) The full-length FhHDM-1 cDNA, minus the N-terminal signal peptide, was expressed in E. coli and the His-tagged recombinant was purified from cell lysates using Ni-NTA agarose (Qiagen). P, pre-column; FT, flow-through; W, wash, E1, imidazole eluate. Co-eluting proteins were removed by RP-HLPC resulting in recombinant FhHDM-1 of very high purity (E2). (B) CD spectra of recombinant 0.1 mg/mL −1 FhHDM-1 at pH 7.3. The wavelength scan was performed between 190 and 250 nm. The final spectrum (closed circles in the absence of 30% (v/v) TFE and open circles in the presence of 30% (v/v) TFE) is the average result from three scans measured at 20°C. The CONTINLL algorithm from the CDPro software package [27] produced the best fit (solid lines) against the SP29 protein database [28] with r.m.s.d. values for all samples ≤0.325. FhHDM-1 adopts a near identical solution structure in both native and recombinant form at both pH 4.5 and pH 7.3 (data not shown). The resulting secondary structure proportions are reported in Table S1 .
    Figure Legend Snippet: Expression and CD spectroscopy of recombinant FhHDM-1. (A) The full-length FhHDM-1 cDNA, minus the N-terminal signal peptide, was expressed in E. coli and the His-tagged recombinant was purified from cell lysates using Ni-NTA agarose (Qiagen). P, pre-column; FT, flow-through; W, wash, E1, imidazole eluate. Co-eluting proteins were removed by RP-HLPC resulting in recombinant FhHDM-1 of very high purity (E2). (B) CD spectra of recombinant 0.1 mg/mL −1 FhHDM-1 at pH 7.3. The wavelength scan was performed between 190 and 250 nm. The final spectrum (closed circles in the absence of 30% (v/v) TFE and open circles in the presence of 30% (v/v) TFE) is the average result from three scans measured at 20°C. The CONTINLL algorithm from the CDPro software package [27] produced the best fit (solid lines) against the SP29 protein database [28] with r.m.s.d. values for all samples ≤0.325. FhHDM-1 adopts a near identical solution structure in both native and recombinant form at both pH 4.5 and pH 7.3 (data not shown). The resulting secondary structure proportions are reported in Table S1 .

    Techniques Used: Expressing, Spectroscopy, Recombinant, Purification, Flow Cytometry, Software, Produced

    9) Product Images from "KLF9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells"

    Article Title: KLF9, a differentiation-associated transcription factor, suppresses Notch1 signaling and inhibits glioblastoma-initiating stem cells

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1002/stem.561

    KLF9 induction inhibits the in growth of xenografts derived from GBM neurospheres
    Figure Legend Snippet: KLF9 induction inhibits the in growth of xenografts derived from GBM neurospheres

    Techniques Used: Derivative Assay

    KLF9 binds to the Notch1 promoter basic transcriptional element (BTE) sites and inhibits promoter activity
    Figure Legend Snippet: KLF9 binds to the Notch1 promoter basic transcriptional element (BTE) sites and inhibits promoter activity

    Techniques Used: Activity Assay

    KLF9 knockdown rescues neurospheres from RA-induced effects
    Figure Legend Snippet: KLF9 knockdown rescues neurospheres from RA-induced effects

    Techniques Used:

    Forced KLF9 expression inhibits neurosphere formation and colony formation
    Figure Legend Snippet: Forced KLF9 expression inhibits neurosphere formation and colony formation

    Techniques Used: Expressing

    Forced KLF9 expression induces cell differentiation and reduces stem-cell marker expression
    Figure Legend Snippet: Forced KLF9 expression induces cell differentiation and reduces stem-cell marker expression

    Techniques Used: Expressing, Cell Differentiation, Marker

    KLF9 suppresses Notch1 transcription in GBM-derived neurospheres
    Figure Legend Snippet: KLF9 suppresses Notch1 transcription in GBM-derived neurospheres

    Techniques Used: Derivative Assay

    Forced differentiation with either RA or serum induces KLF9 expression in GBM-derived neurospheres
    Figure Legend Snippet: Forced differentiation with either RA or serum induces KLF9 expression in GBM-derived neurospheres

    Techniques Used: Expressing, Derivative Assay

    10) Product Images from "Follistatin like-1 (Fstl1) is required for the normal formation of lung airway and vascular smooth muscle at birth"

    Article Title: Follistatin like-1 (Fstl1) is required for the normal formation of lung airway and vascular smooth muscle at birth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177899

    Loss of Fstl1 led to abnormal tracheal and bronchial SM formation in E18.5 embryos. ( A , B ) α-SMA whole-mount staining revealed an extremely attenuated α-SMA signal in Fstl1 −/− trachea. Trachea ( C , D ), proximal bronchi ( E , F , the sections at the points where the tracheas split into the left and right main bronchi) and distal bronchi ( G , H ) sections stained for α-SMA confirmed reduced α-SMA expression (arrows). ( I , J ) Trachea sections of similar planes, as indicated by the common carotid artery and thymus, stained for SM22α revealed reduced SM cells in Fstl1 −/− trachea. ( K , L ) Stitched images showed airway SM defects from proximal bronchi to distal bronchi in Fstl1 −/− lung. ( M ) qRT-PCR analysis of the expression of Fstl1 and α-SMA in E18.5 tracheas and lungs (n = 5 per group). aa, arch of the aorta, th, thymus, ca, common carotid artery, lb, left main bronchus, rb, right main bronchus. *, P
    Figure Legend Snippet: Loss of Fstl1 led to abnormal tracheal and bronchial SM formation in E18.5 embryos. ( A , B ) α-SMA whole-mount staining revealed an extremely attenuated α-SMA signal in Fstl1 −/− trachea. Trachea ( C , D ), proximal bronchi ( E , F , the sections at the points where the tracheas split into the left and right main bronchi) and distal bronchi ( G , H ) sections stained for α-SMA confirmed reduced α-SMA expression (arrows). ( I , J ) Trachea sections of similar planes, as indicated by the common carotid artery and thymus, stained for SM22α revealed reduced SM cells in Fstl1 −/− trachea. ( K , L ) Stitched images showed airway SM defects from proximal bronchi to distal bronchi in Fstl1 −/− lung. ( M ) qRT-PCR analysis of the expression of Fstl1 and α-SMA in E18.5 tracheas and lungs (n = 5 per group). aa, arch of the aorta, th, thymus, ca, common carotid artery, lb, left main bronchus, rb, right main bronchus. *, P

    Techniques Used: Staining, Expressing, Quantitative RT-PCR

    ASM differentiation was significantly reduced in Fstl1 −/− lungs. ( A , B ) α-SMA immunostaining of E10.5 trachea sections revealed rare SM cell differentiation in both WT and Fstl1 −/− (arrows). Immunofluorescence staining for α-SMA of E11.5 ( C , D ), E12.5 ( E , F ), E13.5 ( G , H ), E15.5 ( I , J ) tracheas showed less SM formation and expansion in Fstl1 −/− tracheas during the early development (arrows). ( K ) qRT-PCR of Fstl1 , α-SMA , myocardin and SRF expression demonstrated a significant reduction in E11.5 Fstl1 −/− lungs compared to control WT lungs (WT, n = 5, Fstl1 −/− , n = 6). ( L ) Loss of Fstl1 inhibited the TGF-β1-induced α-SMA , SRF , and myocardin expression in MEFs. *, P
    Figure Legend Snippet: ASM differentiation was significantly reduced in Fstl1 −/− lungs. ( A , B ) α-SMA immunostaining of E10.5 trachea sections revealed rare SM cell differentiation in both WT and Fstl1 −/− (arrows). Immunofluorescence staining for α-SMA of E11.5 ( C , D ), E12.5 ( E , F ), E13.5 ( G , H ), E15.5 ( I , J ) tracheas showed less SM formation and expansion in Fstl1 −/− tracheas during the early development (arrows). ( K ) qRT-PCR of Fstl1 , α-SMA , myocardin and SRF expression demonstrated a significant reduction in E11.5 Fstl1 −/− lungs compared to control WT lungs (WT, n = 5, Fstl1 −/− , n = 6). ( L ) Loss of Fstl1 inhibited the TGF-β1-induced α-SMA , SRF , and myocardin expression in MEFs. *, P

    Techniques Used: Immunostaining, Cell Differentiation, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

    11) Product Images from "ToxR Recognizes a Direct Repeat Element in the toxT, ompU, ompT, and ctxA Promoters of Vibrio cholerae To Regulate Transcription"

    Article Title: ToxR Recognizes a Direct Repeat Element in the toxT, ompU, ompT, and ctxA Promoters of Vibrio cholerae To Regulate Transcription

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00889-12

    Models for the role of ToxR in TcpP-mediated toxT activation. (A) As previously described, the toxT ). (B) In the “hand-holding” model, ToxR and TcpP interact in the inner membrane of V. cholerae ), and then ToxR escorts TcpP to the toxT promoter where ToxR relieves H-NS repression and maintains interaction with TcpP while TcpP stimulates transcription. (C) In the “catch and release” model, ToxR also interacts with TcpP and recruits TcpP to the toxT ). (D) In the “promoter alteration” model, interaction between ToxR and TcpP is not required for toxT activation; rather, ToxR binding to the toxT promoter displaces H-NS and alters the toxT promoter architecture such that a normally weak TcpP-binding site is altered in some way to facilitate enhanced TcpP binding, thus allowing TcpP-mediated activation of the toxT promoter. (E) In the “membrane recruitment” model, again interaction between ToxR and TcpP is not required, but the role of ToxR is to simply recruit the toxT promoter to the membrane where TcpP has easier access to its DNA-binding site. This model takes into account the fact that TcpP binding to the toxT promoter requires higher concentrations of V. cholerae ) and the fact that membrane localization was previously shown to be required for ToxR to facilitate TcpP-mediated toxT ).
    Figure Legend Snippet: Models for the role of ToxR in TcpP-mediated toxT activation. (A) As previously described, the toxT ). (B) In the “hand-holding” model, ToxR and TcpP interact in the inner membrane of V. cholerae ), and then ToxR escorts TcpP to the toxT promoter where ToxR relieves H-NS repression and maintains interaction with TcpP while TcpP stimulates transcription. (C) In the “catch and release” model, ToxR also interacts with TcpP and recruits TcpP to the toxT ). (D) In the “promoter alteration” model, interaction between ToxR and TcpP is not required for toxT activation; rather, ToxR binding to the toxT promoter displaces H-NS and alters the toxT promoter architecture such that a normally weak TcpP-binding site is altered in some way to facilitate enhanced TcpP binding, thus allowing TcpP-mediated activation of the toxT promoter. (E) In the “membrane recruitment” model, again interaction between ToxR and TcpP is not required, but the role of ToxR is to simply recruit the toxT promoter to the membrane where TcpP has easier access to its DNA-binding site. This model takes into account the fact that TcpP binding to the toxT promoter requires higher concentrations of V. cholerae ) and the fact that membrane localization was previously shown to be required for ToxR to facilitate TcpP-mediated toxT ).

    Techniques Used: Activation Assay, Binding Assay

    ToxR fails to bind or activate a toxT-lacZ derivative containing the degenerate ToxR-binding site from −69 to −56. (A) toxT promoter derivatives driving lacZ expression were tested for activation in wild-type V. cholerae (O395) or the toxR mutant strain EK307 with or without overexpression of ToxRS from plasmid pVJ21. n = 6. (B) Electrophoretic mobility shift analysis of full-length (−172 to +45), −100 to +45, −81 to +45, and −46 to + 45 toxT derivatives with increasing concentrations of ToxR-containing membranes shows the degenerate ToxR-binding site from −69 to −56 has weak ToxR binding capacity. Negative-control gel shifting with membranes lacking ToxR (ToxR − ) was also tested and showed minimal background. DNA bound by membrane-localized ToxR is retained in the well of the gel. The percentage of shifting by membranes is indicated under each lane as determined by ImageJ.
    Figure Legend Snippet: ToxR fails to bind or activate a toxT-lacZ derivative containing the degenerate ToxR-binding site from −69 to −56. (A) toxT promoter derivatives driving lacZ expression were tested for activation in wild-type V. cholerae (O395) or the toxR mutant strain EK307 with or without overexpression of ToxRS from plasmid pVJ21. n = 6. (B) Electrophoretic mobility shift analysis of full-length (−172 to +45), −100 to +45, −81 to +45, and −46 to + 45 toxT derivatives with increasing concentrations of ToxR-containing membranes shows the degenerate ToxR-binding site from −69 to −56 has weak ToxR binding capacity. Negative-control gel shifting with membranes lacking ToxR (ToxR − ) was also tested and showed minimal background. DNA bound by membrane-localized ToxR is retained in the well of the gel. The percentage of shifting by membranes is indicated under each lane as determined by ImageJ.

    Techniques Used: Binding Assay, Expressing, Activation Assay, Mutagenesis, Over Expression, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Negative Control

    DNA sequence of the V. cholerae classical strain O395 promoter-proximal region of the toxT promoter and ToxR-dependent activation of single-base-pair substitutions. (A) Nucleotides are numbered relative to the toxT ). The solid gray arrows above the sequence indicate the position of the putative 5′-TNAAA-N 5 ). Single-nucleotide substitutions generated within the toxT promoter region from −100 to −57 are indicated on the bottom line in italics. (B) Effects of ToxR-binding site mutations on toxT-lacZ activity in wild-type V. cholerae strain O395. Strains carrying a plasmid-borne wild-type toxT-lacZ fusion (−172 to +45), single-base-pair substitution toxT promoter mutants, promoter deletion derivatives, or empty vector (promoterless lacZ vector, pTG24) were assessed for β-galactosidase activity. The positions of substitutions and endpoints are indicated relative to the toxT transcription start site. Error bars represent the standard deviations for each data set. *, P
    Figure Legend Snippet: DNA sequence of the V. cholerae classical strain O395 promoter-proximal region of the toxT promoter and ToxR-dependent activation of single-base-pair substitutions. (A) Nucleotides are numbered relative to the toxT ). The solid gray arrows above the sequence indicate the position of the putative 5′-TNAAA-N 5 ). Single-nucleotide substitutions generated within the toxT promoter region from −100 to −57 are indicated on the bottom line in italics. (B) Effects of ToxR-binding site mutations on toxT-lacZ activity in wild-type V. cholerae strain O395. Strains carrying a plasmid-borne wild-type toxT-lacZ fusion (−172 to +45), single-base-pair substitution toxT promoter mutants, promoter deletion derivatives, or empty vector (promoterless lacZ vector, pTG24) were assessed for β-galactosidase activity. The positions of substitutions and endpoints are indicated relative to the toxT transcription start site. Error bars represent the standard deviations for each data set. *, P

    Techniques Used: Sequencing, Activation Assay, Generated, Binding Assay, Activity Assay, Plasmid Preparation

    The ToxR consensus-binding site is required for ToxR-mediated activation of the ompU and ctxA promoters and repression of the ompT promoter. (A) Location of consensus ToxR-binding sites in the ompU , ctxA , and ompT promoters. Nucleotides comprising potential ToxR-binding sites are in bold, while the opposite strand sequences, matching the toxT promoter consensus ToxR-binding site, are shown in gray. Those nucleotides targeted for mutagenesis are highlighted in gray and underlined. (B) Effects of transversion mutations on ToxR-mediated activation of the ompU promoter in wild-type V. cholerae or the toxR mutant strain, EK307. (C) Effect of mutations in the consensus ToxR-binding site within the promoter-proximal heptad repeat of the ctxA promoter. ctxA-lacZ expression was measured in a Δ toxT strain (ToxR dependent) or wild-type V. cholerae O395 (ToxT dependent). (D) Effects of ompT transversion mutations on ToxR-mediated repression of the ompT promoter in wild-type V. cholerae or the toxR mutant strain, EK307. *, P
    Figure Legend Snippet: The ToxR consensus-binding site is required for ToxR-mediated activation of the ompU and ctxA promoters and repression of the ompT promoter. (A) Location of consensus ToxR-binding sites in the ompU , ctxA , and ompT promoters. Nucleotides comprising potential ToxR-binding sites are in bold, while the opposite strand sequences, matching the toxT promoter consensus ToxR-binding site, are shown in gray. Those nucleotides targeted for mutagenesis are highlighted in gray and underlined. (B) Effects of transversion mutations on ToxR-mediated activation of the ompU promoter in wild-type V. cholerae or the toxR mutant strain, EK307. (C) Effect of mutations in the consensus ToxR-binding site within the promoter-proximal heptad repeat of the ctxA promoter. ctxA-lacZ expression was measured in a Δ toxT strain (ToxR dependent) or wild-type V. cholerae O395 (ToxT dependent). (D) Effects of ompT transversion mutations on ToxR-mediated repression of the ompT promoter in wild-type V. cholerae or the toxR mutant strain, EK307. *, P

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

    12) Product Images from "Acute and Persistent Infection of Human Neural Cell Lines by Human Coronavirus OC43"

    Article Title: Acute and Persistent Infection of Human Neural Cell Lines by Human Coronavirus OC43

    Journal: Journal of Virology

    doi:

    Detection of human coronavirus antigens by indirect immunofluorescence on cells acutely infected by HuCV-OC43, using virus-specific MAb (1.10C.3). (A) H4 cells; (B) SK-N-SH cells; (C) U-373 MG cells; (D) U-87 MG cells; (E) GL-15 cells; (F) MO3.13 cells; (G) CHME-5 cells; (H) HRT-18 cells.
    Figure Legend Snippet: Detection of human coronavirus antigens by indirect immunofluorescence on cells acutely infected by HuCV-OC43, using virus-specific MAb (1.10C.3). (A) H4 cells; (B) SK-N-SH cells; (C) U-373 MG cells; (D) U-87 MG cells; (E) GL-15 cells; (F) MO3.13 cells; (G) CHME-5 cells; (H) HRT-18 cells.

    Techniques Used: Immunofluorescence, Infection

    Detection of HuCV antigens by indirect immunofluorescence on cells persistently infected by HuCV-OC43, using virus-specific MAb (1.10C.3). (A) H4 cells, passage 40; (B) MO3.13 cells, passage 21; (C) U-373 MG cells, passage 22; (D) U-87 MG cells, passage 24; (E) HRT-18 cells, passage 40; (F) isotypic control MAb (5-11H.6) on virus-infected U-373 MG cells.
    Figure Legend Snippet: Detection of HuCV antigens by indirect immunofluorescence on cells persistently infected by HuCV-OC43, using virus-specific MAb (1.10C.3). (A) H4 cells, passage 40; (B) MO3.13 cells, passage 21; (C) U-373 MG cells, passage 22; (D) U-87 MG cells, passage 24; (E) HRT-18 cells, passage 40; (F) isotypic control MAb (5-11H.6) on virus-infected U-373 MG cells.

    Techniques Used: Immunofluorescence, Infection

    Cytopathic effects of a persistent HuCV-OC43 infection on MO3.13 cells. (A) Noninfected cells; (B) HuCV-OC43-infected MO3.13 cells, passage 5.
    Figure Legend Snippet: Cytopathic effects of a persistent HuCV-OC43 infection on MO3.13 cells. (A) Noninfected cells; (B) HuCV-OC43-infected MO3.13 cells, passage 5.

    Techniques Used: Infection

    13) Product Images from "Effect of Intragenic Rearrangement and Changes in the 3? Consensus Sequence on NSP1 Expression and Rotavirus Replication"

    Article Title: Effect of Intragenic Rearrangement and Changes in the 3? Consensus Sequence on NSP1 Expression and Rotavirus Replication

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.5.2076-2086.2001

    Effect of the UGACC→UGAACC mutation on protein expression in infected cells. The chimeric reporter RNAs g6-Fluc-UGACC and -UGAACC and glo/g6-Fluc-UGACC and -UGAACC were separately cotransfected with nv-Rluc into SA11-infected MA104 cells at 1 h p.i. At 9 h p.i., the levels of firefly and Renilla luciferases per milligram of cell lysate were determined and the expression of firefly luciferase was normalized to the expression of Renilla luciferase. To ease the comparison of values, the expression of firefly luciferase for the g6-Fluc-UGACC and glo/g6-Fluc-UGACC RNAs was set to 100%.
    Figure Legend Snippet: Effect of the UGACC→UGAACC mutation on protein expression in infected cells. The chimeric reporter RNAs g6-Fluc-UGACC and -UGAACC and glo/g6-Fluc-UGACC and -UGAACC were separately cotransfected with nv-Rluc into SA11-infected MA104 cells at 1 h p.i. At 9 h p.i., the levels of firefly and Renilla luciferases per milligram of cell lysate were determined and the expression of firefly luciferase was normalized to the expression of Renilla luciferase. To ease the comparison of values, the expression of firefly luciferase for the g6-Fluc-UGACC and glo/g6-Fluc-UGACC RNAs was set to 100%.

    Techniques Used: Mutagenesis, Expressing, Infection, Luciferase

    14) Product Images from "Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid"

    Article Title: Molecular cloning and characterization of a novel tomato xylosyltransferase specific for gentisic acid

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erq234

    Xylosyltransferase activity of recombinant GAGT. Left panel: GAGT cDNA was expressed in Pichia pastoris cells under the control of a methanol-inducible promoter (see the Materials and methods). Extracts from the uninduced (lane 1) and methanol-induced cells (lane 2) were incubated with UDP[ 14 C]-xylose and GA. Lane 3 corresponds to UDP[ 14 C]-xylose. Right panel: samples of UDP-xylose (lane 1), standard GA-5- O -β- D -xyloside, previously obtained in our laboratory (lane 2; Fayos et al. , 2006 ) and the GA xyloside produced using the crude tomato leaf extracts (lane 3) were separated by TLC under the same conditions, and were chemically revealed as described in the text.
    Figure Legend Snippet: Xylosyltransferase activity of recombinant GAGT. Left panel: GAGT cDNA was expressed in Pichia pastoris cells under the control of a methanol-inducible promoter (see the Materials and methods). Extracts from the uninduced (lane 1) and methanol-induced cells (lane 2) were incubated with UDP[ 14 C]-xylose and GA. Lane 3 corresponds to UDP[ 14 C]-xylose. Right panel: samples of UDP-xylose (lane 1), standard GA-5- O -β- D -xyloside, previously obtained in our laboratory (lane 2; Fayos et al. , 2006 ) and the GA xyloside produced using the crude tomato leaf extracts (lane 3) were separated by TLC under the same conditions, and were chemically revealed as described in the text.

    Techniques Used: Activity Assay, Recombinant, Incubation, Produced, Thin Layer Chromatography

    15) Product Images from "Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare"

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00443

    Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
    Figure Legend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

    Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.
    Figure Legend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Techniques Used: Polymerase Chain Reaction, Marker, Mouse Assay

    16) Product Images from "Detection of Severe Acute Respiratory Syndrome Coronavirus in Blood of Infected Patients"

    Article Title: Detection of Severe Acute Respiratory Syndrome Coronavirus in Blood of Infected Patients

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.42.1.347-350.2004

    Detection of SARS CoV from 18 subjects by real-time quantitative PCR. Data for RNA from 18 subjects are shown. The viral load is indicated as number of copies per ml of blood. Subject 1, 2,020 copies; subject 2, 9,100 copies; subject 3, 3,120 copies; subject 4, 9,600 copies. A sample would be considered positive if it generated a typical amplification curve within the 45 cycles. Negative signals from subjects 5 to 18 and the nontemplate control (water) are shown. The inset graph shows melting-curve analysis of the PCR products from the 18 subjects. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level.
    Figure Legend Snippet: Detection of SARS CoV from 18 subjects by real-time quantitative PCR. Data for RNA from 18 subjects are shown. The viral load is indicated as number of copies per ml of blood. Subject 1, 2,020 copies; subject 2, 9,100 copies; subject 3, 3,120 copies; subject 4, 9,600 copies. A sample would be considered positive if it generated a typical amplification curve within the 45 cycles. Negative signals from subjects 5 to 18 and the nontemplate control (water) are shown. The inset graph shows melting-curve analysis of the PCR products from the 18 subjects. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level.

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

    Detection of SARS CoV by real-time quantitative PCR. (A) Amplification of single-stranded RNA standards (indicated as a to e) and RNA extracted from sputum, stool, and blood spiked with virus grown in Vero E6 cells. The x axis denotes the cycle number of the quantitative PCR assay, and the y axis denotes fluorescence intensity (F2) over the background level. The nontemplate control (water) is indicated. The viral load is indicated as the number of copies per reaction: spiked sputum, 5 × 10 3 ; spiked stool, 4 × 10 3 ; spiked blood, 1 × 10 3 . RNA standards were as follows: (a) 1 × 10 6 copies per reaction, (b) 9.5 × 10 4 copies per reaction, (c) 8.7 × 10 3 copies per reaction, (d) 1.1 × 10 3 copies per reaction, and (e) 8.5 × 10 copies per reaction. The inset graph represents melting-curve analysis of the PCR products. Signals from RNA standards (a to e), spiked samples, normal samples, and nontemplate control (water) are shown. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level. (B) Detection of the internal control in fluorometer channel F3 in parallel with the simultaneous amplification of the RNA standards (a to e), spiked samples, and nontemplate control is shown.
    Figure Legend Snippet: Detection of SARS CoV by real-time quantitative PCR. (A) Amplification of single-stranded RNA standards (indicated as a to e) and RNA extracted from sputum, stool, and blood spiked with virus grown in Vero E6 cells. The x axis denotes the cycle number of the quantitative PCR assay, and the y axis denotes fluorescence intensity (F2) over the background level. The nontemplate control (water) is indicated. The viral load is indicated as the number of copies per reaction: spiked sputum, 5 × 10 3 ; spiked stool, 4 × 10 3 ; spiked blood, 1 × 10 3 . RNA standards were as follows: (a) 1 × 10 6 copies per reaction, (b) 9.5 × 10 4 copies per reaction, (c) 8.7 × 10 3 copies per reaction, (d) 1.1 × 10 3 copies per reaction, and (e) 8.5 × 10 copies per reaction. The inset graph represents melting-curve analysis of the PCR products. Signals from RNA standards (a to e), spiked samples, normal samples, and nontemplate control (water) are shown. The x axis denotes the temperature ( o C), and the y axis denotes the fluorescence intensity (F2) over the background level. (B) Detection of the internal control in fluorometer channel F3 in parallel with the simultaneous amplification of the RNA standards (a to e), spiked samples, and nontemplate control is shown.

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

    17) Product Images from "Recurrent (2;2) and (2;8) Translocations in Rhabdomyosarcoma without the Canonical PAX-FOXO1 fuse PAX3 to Members of the Nuclear Receptor Transcriptional Coactivator (NCOA) Family"

    Article Title: Recurrent (2;2) and (2;8) Translocations in Rhabdomyosarcoma without the Canonical PAX-FOXO1 fuse PAX3 to Members of the Nuclear Receptor Transcriptional Coactivator (NCOA) Family

    Journal: Genes, chromosomes & cancer

    doi: 10.1002/gcc.20731

    Representative FISH analysis of t(2;2)(p23;q35) and t(2;8)(q35;q12) translocations. A. FISH analysis of Case 1 with the custom designed NCOA1 break-apart probe set demonstrates split orange and green signals (arrows) indicative of a rearrangement of this locus. B. FISH analysis of Case 1 with the PAX3 spanning probe set in orange and the NCOA1 spanning probe set in green demonstrates the presence of juxtaposed or fused orange and green signals consistent with the RT-PCR findings of a PAX3-NCOA1 fusion transcript in this case. C and D. FISH analyses of Case 4 with the custom designed PAX3 and NCOA2 break-apart probe sets, respectively, demonstrate split orange and green signals (arrows) indicative of a rearrangement of each of these loci.
    Figure Legend Snippet: Representative FISH analysis of t(2;2)(p23;q35) and t(2;8)(q35;q12) translocations. A. FISH analysis of Case 1 with the custom designed NCOA1 break-apart probe set demonstrates split orange and green signals (arrows) indicative of a rearrangement of this locus. B. FISH analysis of Case 1 with the PAX3 spanning probe set in orange and the NCOA1 spanning probe set in green demonstrates the presence of juxtaposed or fused orange and green signals consistent with the RT-PCR findings of a PAX3-NCOA1 fusion transcript in this case. C and D. FISH analyses of Case 4 with the custom designed PAX3 and NCOA2 break-apart probe sets, respectively, demonstrate split orange and green signals (arrows) indicative of a rearrangement of each of these loci.

    Techniques Used: Fluorescence In Situ Hybridization, Reverse Transcription Polymerase Chain Reaction

    Effect of PAX3-NCOA1 type 1, PAX3-NCOA1 type 2 and PAX3-NCOA2 expression on NIH3T3 cell growth. A. NIH3T3-Tet-19.1 cells transfected with plasmids pcDNA4/TO (1), PAX3-NCOA1-type 1 (2), PAX3-NCOA1-type 2 (3) and PAX3-NCOA2 (4) in pcDNA4/TO and grown in the presence of 1 μg/ml tetracycline. B. Representative agar assay images acquired 2 weeks after cells were plated at low density and grown in the presence of 1 μg/ml tetracycline show formation of macroscopic colonies in the PAX3-NCOA1-type 1 (2), PAX3-NCOA1-type 2 (3) and PAX3-NCOA2 (4) plates but not in the pcDNA4/TO (1) plate.
    Figure Legend Snippet: Effect of PAX3-NCOA1 type 1, PAX3-NCOA1 type 2 and PAX3-NCOA2 expression on NIH3T3 cell growth. A. NIH3T3-Tet-19.1 cells transfected with plasmids pcDNA4/TO (1), PAX3-NCOA1-type 1 (2), PAX3-NCOA1-type 2 (3) and PAX3-NCOA2 (4) in pcDNA4/TO and grown in the presence of 1 μg/ml tetracycline. B. Representative agar assay images acquired 2 weeks after cells were plated at low density and grown in the presence of 1 μg/ml tetracycline show formation of macroscopic colonies in the PAX3-NCOA1-type 1 (2), PAX3-NCOA1-type 2 (3) and PAX3-NCOA2 (4) plates but not in the pcDNA4/TO (1) plate.

    Techniques Used: Expressing, Transfection

    Tetracycline inducible expression of PAX3-NCOA1 and PAX3-NCOA2 genes in transfected NIH 3T3 cells. A . Representative Western blots demonstrate the induction of varying expression levels of PAX3-NCOA1 type 1, PAX3-NCOA1 type 2 and PAX3-NCOA2 chimeric genes that correspond with different doses of tetracycline. B. Western blot demonstrating the rapid induction of PAX3-NCOA1 type 1 expression in the presence of 2.5 μg/ml tetracycline (cells were harvested at the time points indicated).
    Figure Legend Snippet: Tetracycline inducible expression of PAX3-NCOA1 and PAX3-NCOA2 genes in transfected NIH 3T3 cells. A . Representative Western blots demonstrate the induction of varying expression levels of PAX3-NCOA1 type 1, PAX3-NCOA1 type 2 and PAX3-NCOA2 chimeric genes that correspond with different doses of tetracycline. B. Western blot demonstrating the rapid induction of PAX3-NCOA1 type 1 expression in the presence of 2.5 μg/ml tetracycline (cells were harvested at the time points indicated).

    Techniques Used: Expressing, Transfection, Western Blot

    Analyses of transforming activities of PAX3-NCOA1 and PAX3-NCOA2 fusion proteins and deletion mutants. A. Schematic structure of PAX3-NCOA1, PAX3-NCOA2 and deletion mutants. Solid bars with amino acid position displays the regions deleted. B. Immunoblot analyses of PAX3-NCOA1, PAX3-NCOA2 and deletion mutants in NIH3T3-Tet-19.1.E. C. Soft agar colony assay of pcDNA4/TO vector (1), pcDNA4/TO-PAX3-NCOA1 type 1, pcDNA4/TO-PAX3-NCOA1 type 2, and pcDNA4/TO-PAX3-NCOA2 transduced NIH3T3-Tet-19.1 cells. Cells were incubated in the presence of 1μg/ml tetracycline and scored after 14 days. Three plates were counted for each construct.
    Figure Legend Snippet: Analyses of transforming activities of PAX3-NCOA1 and PAX3-NCOA2 fusion proteins and deletion mutants. A. Schematic structure of PAX3-NCOA1, PAX3-NCOA2 and deletion mutants. Solid bars with amino acid position displays the regions deleted. B. Immunoblot analyses of PAX3-NCOA1, PAX3-NCOA2 and deletion mutants in NIH3T3-Tet-19.1.E. C. Soft agar colony assay of pcDNA4/TO vector (1), pcDNA4/TO-PAX3-NCOA1 type 1, pcDNA4/TO-PAX3-NCOA1 type 2, and pcDNA4/TO-PAX3-NCOA2 transduced NIH3T3-Tet-19.1 cells. Cells were incubated in the presence of 1μg/ml tetracycline and scored after 14 days. Three plates were counted for each construct.

    Techniques Used: Colony Assay, Plasmid Preparation, Incubation, Construct

    Comparison of wild type and fusion products associated with the t(2;2)(p23;q35) translocation. A-C. Genomic structure of PAX3, NCOA1 , PAX3-NCOA1 fusion type 1 ( PAX3 exons 1-6 and NCOA1 exons 12-20), and PAX3-NCOA1 type 2 fusion ( PAX3 exons 1-7 and NCOA1 exons 11-20), D. Structure of the proteins involved in the fusion. Interacting proteins are displayed as bars. The letters within the bars designate conserved domains (PB, paired domain; HD, homeodomain of the PAX3 protein; and, bHLH/PAS, receptor nuclear translocator domain, involved in DNA binding). S/T represents the serine-threonine-rich region. Transcriptional domains of PAX3 are DBD, DNA binding domain and TAD, transcriptional activation domain. The two transcriptional activator domains of NCOA1 are CID/AD1 and AD2. Encircled L4, and L5 are atypical LXXLL a-helix motifs and boxed L6 is an atypical LXXLL motif, HAT indicates the histone acetyltransferase domain. Factors that interact with specific functional domains are listed beneath the lines of the corresponding domain bars.
    Figure Legend Snippet: Comparison of wild type and fusion products associated with the t(2;2)(p23;q35) translocation. A-C. Genomic structure of PAX3, NCOA1 , PAX3-NCOA1 fusion type 1 ( PAX3 exons 1-6 and NCOA1 exons 12-20), and PAX3-NCOA1 type 2 fusion ( PAX3 exons 1-7 and NCOA1 exons 11-20), D. Structure of the proteins involved in the fusion. Interacting proteins are displayed as bars. The letters within the bars designate conserved domains (PB, paired domain; HD, homeodomain of the PAX3 protein; and, bHLH/PAS, receptor nuclear translocator domain, involved in DNA binding). S/T represents the serine-threonine-rich region. Transcriptional domains of PAX3 are DBD, DNA binding domain and TAD, transcriptional activation domain. The two transcriptional activator domains of NCOA1 are CID/AD1 and AD2. Encircled L4, and L5 are atypical LXXLL a-helix motifs and boxed L6 is an atypical LXXLL motif, HAT indicates the histone acetyltransferase domain. Factors that interact with specific functional domains are listed beneath the lines of the corresponding domain bars.

    Techniques Used: Translocation Assay, Binding Assay, Activation Assay, HAT Assay, Functional Assay

    Representative RT-PCR and sequence analyses for chimeric transcripts in Cases 1-5. A. Detection of PAX3-NCOA1 transcripts in Case 1 (lanes 1 and 2); 2 (lanes 3 and 4) and 3 (lanes 5 and 6). Primers used for lanes 1, 3 and 5 were PAX3-32 and NCOA1-33; for lanes 2, 4 and 6, PAX3-34 and NCOA1-35. Detection of PAX3-NCOA2 transcripts in Cases 4 (lane 7) and 5 (lane 8). Primers used were PAX3-41 and NCOA2-48. B . Sequence alignment of the PAX3-NCOA1 and PAX3-NCOA2 breakpoint regions. Arrows depict the fusion point. Single letter amino acid code is displayed beneath the nucleotide sequence.
    Figure Legend Snippet: Representative RT-PCR and sequence analyses for chimeric transcripts in Cases 1-5. A. Detection of PAX3-NCOA1 transcripts in Case 1 (lanes 1 and 2); 2 (lanes 3 and 4) and 3 (lanes 5 and 6). Primers used for lanes 1, 3 and 5 were PAX3-32 and NCOA1-33; for lanes 2, 4 and 6, PAX3-34 and NCOA1-35. Detection of PAX3-NCOA2 transcripts in Cases 4 (lane 7) and 5 (lane 8). Primers used were PAX3-41 and NCOA2-48. B . Sequence alignment of the PAX3-NCOA1 and PAX3-NCOA2 breakpoint regions. Arrows depict the fusion point. Single letter amino acid code is displayed beneath the nucleotide sequence.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

    18) Product Images from "Shiga Toxin 2 and Flagellin from Shiga-Toxigenic Escherichia coli Superinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation ▿"

    Article Title: Shiga Toxin 2 and Flagellin from Shiga-Toxigenic Escherichia coli Superinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00383-10

    Superinduction of flagellin-mediated IL-8 mRNA in HCT-8 cells by Stx2. HCT-8 cells were treated with 100 ng/ml rFliC H21 (FliC) or HI rFliC H21 (HI FliC), with or without 1 μg/ml Stx2, for 1 or 4 h, and then total RNA was extracted and IL-8 mRNA
    Figure Legend Snippet: Superinduction of flagellin-mediated IL-8 mRNA in HCT-8 cells by Stx2. HCT-8 cells were treated with 100 ng/ml rFliC H21 (FliC) or HI rFliC H21 (HI FliC), with or without 1 μg/ml Stx2, for 1 or 4 h, and then total RNA was extracted and IL-8 mRNA

    Techniques Used:

    Effect of p38 MAPK inhibition on IL-8 mRNA and protein induction by flagellin. HCT-8 cells were stimulated with 100 ng/ml rFliC H21 in the presence of either 10 μM SB203580 (SB) (p38 inhibitor) or an equal volume of vehicle (DMSO). (A) At 1 or
    Figure Legend Snippet: Effect of p38 MAPK inhibition on IL-8 mRNA and protein induction by flagellin. HCT-8 cells were stimulated with 100 ng/ml rFliC H21 in the presence of either 10 μM SB203580 (SB) (p38 inhibitor) or an equal volume of vehicle (DMSO). (A) At 1 or

    Techniques Used: Inhibition

    19) Product Images from "A large deletion in RPGR causes XLPRA in Weimaraner dogs"

    Article Title: A large deletion in RPGR causes XLPRA in Weimaraner dogs

    Journal: Canine Genetics and Epidemiology

    doi: 10.1186/s40575-016-0037-x

    The deletion in the X chromosomal RPGR gene as identified in a PRA pedigree of Weimaraner dogs via whole exome sequencing; the breakpoint (BP) region is indicated. a Pedigree structure and RPGR deletion genotypes of 18 investigated individuals of the XLPRA Weimaraner family. PRA segregates in two generations of the family. Squares represent males, circles indicate females, crossed-out symbols represent deceased dogs. Filled squares show ophthalmologically diagnosed PRA-affected male dogs. Half-filled circles indicate females with ophthalmologically confirmed mild PRA symptoms. Open symbols represent male and female dogs with normal sight as revealed by general veterinarian examinations, respectively. An asterisk below solid square symbols indicates PRA-diagnosed dogs, which were used for whole exome sequencing. Genotypes of RPGR deletion screening are shown below the symbols. X M (colored in red) refers to an allele with RPGR deletion, X and Y symbols illustrate normal X- (with wildtype RPGR alleles) and Y-chromosomes, respectively. b Integrated Genomics Viewer (IGV) display of the canine RPGR deletion and surrounding regions (CFAX: 3310100–33106500, CanFam3.1 UCSC genome browser) as well as graphical illustration of exon-intron boundaries from the 5′UTR to exon 5. As viewed in IGV, the control and male PRA-affected dog are represented by two separate panels. The upper panel is a histogram where the height of each mountain-like grey area is representative of the read depth at that location. The lower panel is a graphical view of some of the reads that align to that location. Lack of reads (horizontal bars in lower panel) is characteristic for complete loss of exonic sequences. The deletion comprising exons 1–4 (~5 kb) is obvious in the male PRA-affected dog in hemizygous state in contrast to the PRA-unaffected dog. Thus the gap region includes exon 1 in the canine genomic RPGR sequence explaining only non-specific read alignments in the lower panel for the control. c QPCR-based copy number analysis of the deleted exons 3–4 and the non-deleted exon 5 of RPGR gene in four individuals of the pedigree of Weimaraners in comparison to a healthy control. Error bars indicate the standard deviation of three replicates. d Chromatogram and graphical representation of the BP region in the RPGR gene in a male PRA-affected Weimaraner. The graphical illustration indicates part of intron 4 sequence and of 5′UTR of RPGR . Deleted sequences of intron 4 and 5′UTR are coloured in light grey, non-deleted sequences are indicated by coloured letters. The BP region comprises three nucleotides (TTC) from either end which are underlined. The chromatogram also shows the BP (marked with arrows) as well as flanking sequences of intron 4 and 5′UTR of RPGR as identified in a male PRA-affected Weimaraner
    Figure Legend Snippet: The deletion in the X chromosomal RPGR gene as identified in a PRA pedigree of Weimaraner dogs via whole exome sequencing; the breakpoint (BP) region is indicated. a Pedigree structure and RPGR deletion genotypes of 18 investigated individuals of the XLPRA Weimaraner family. PRA segregates in two generations of the family. Squares represent males, circles indicate females, crossed-out symbols represent deceased dogs. Filled squares show ophthalmologically diagnosed PRA-affected male dogs. Half-filled circles indicate females with ophthalmologically confirmed mild PRA symptoms. Open symbols represent male and female dogs with normal sight as revealed by general veterinarian examinations, respectively. An asterisk below solid square symbols indicates PRA-diagnosed dogs, which were used for whole exome sequencing. Genotypes of RPGR deletion screening are shown below the symbols. X M (colored in red) refers to an allele with RPGR deletion, X and Y symbols illustrate normal X- (with wildtype RPGR alleles) and Y-chromosomes, respectively. b Integrated Genomics Viewer (IGV) display of the canine RPGR deletion and surrounding regions (CFAX: 3310100–33106500, CanFam3.1 UCSC genome browser) as well as graphical illustration of exon-intron boundaries from the 5′UTR to exon 5. As viewed in IGV, the control and male PRA-affected dog are represented by two separate panels. The upper panel is a histogram where the height of each mountain-like grey area is representative of the read depth at that location. The lower panel is a graphical view of some of the reads that align to that location. Lack of reads (horizontal bars in lower panel) is characteristic for complete loss of exonic sequences. The deletion comprising exons 1–4 (~5 kb) is obvious in the male PRA-affected dog in hemizygous state in contrast to the PRA-unaffected dog. Thus the gap region includes exon 1 in the canine genomic RPGR sequence explaining only non-specific read alignments in the lower panel for the control. c QPCR-based copy number analysis of the deleted exons 3–4 and the non-deleted exon 5 of RPGR gene in four individuals of the pedigree of Weimaraners in comparison to a healthy control. Error bars indicate the standard deviation of three replicates. d Chromatogram and graphical representation of the BP region in the RPGR gene in a male PRA-affected Weimaraner. The graphical illustration indicates part of intron 4 sequence and of 5′UTR of RPGR . Deleted sequences of intron 4 and 5′UTR are coloured in light grey, non-deleted sequences are indicated by coloured letters. The BP region comprises three nucleotides (TTC) from either end which are underlined. The chromatogram also shows the BP (marked with arrows) as well as flanking sequences of intron 4 and 5′UTR of RPGR as identified in a male PRA-affected Weimaraner

    Techniques Used: Sequencing, Real-time Polymerase Chain Reaction, Standard Deviation

    20) Product Images from "Shiga Toxin 2 and Flagellin from Shiga-Toxigenic Escherichia coli Superinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation ▿"

    Article Title: Shiga Toxin 2 and Flagellin from Shiga-Toxigenic Escherichia coli Superinduce Interleukin-8 through Synergistic Effects on Host Stress-Activated Protein Kinase Activation ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00383-10

    Superinduction of flagellin-mediated IL-8 mRNA in HCT-8 cells by Stx2. HCT-8 cells were treated with 100 ng/ml rFliC H21 (FliC) or HI rFliC H21 (HI FliC), with or without 1 μg/ml Stx2, for 1 or 4 h, and then total RNA was extracted and IL-8 mRNA
    Figure Legend Snippet: Superinduction of flagellin-mediated IL-8 mRNA in HCT-8 cells by Stx2. HCT-8 cells were treated with 100 ng/ml rFliC H21 (FliC) or HI rFliC H21 (HI FliC), with or without 1 μg/ml Stx2, for 1 or 4 h, and then total RNA was extracted and IL-8 mRNA

    Techniques Used:

    Effect of p38 MAPK inhibition on IL-8 mRNA and protein induction by flagellin. HCT-8 cells were stimulated with 100 ng/ml rFliC H21 in the presence of either 10 μM SB203580 (SB) (p38 inhibitor) or an equal volume of vehicle (DMSO). (A) At 1 or
    Figure Legend Snippet: Effect of p38 MAPK inhibition on IL-8 mRNA and protein induction by flagellin. HCT-8 cells were stimulated with 100 ng/ml rFliC H21 in the presence of either 10 μM SB203580 (SB) (p38 inhibitor) or an equal volume of vehicle (DMSO). (A) At 1 or

    Techniques Used: Inhibition

    21) Product Images from "Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis"

    Article Title: Persistence of infectious hepadnavirus in the offspring of woodchuck mothers recovered from viral hepatitis

    Journal: Journal of Clinical Investigation

    doi:

    Detection of WHV cccDNA in selected WHV DNA–reactive liver, PBMCs, and lymphoid tissue samples from 3B/M, 4B/M, and 5C/F offspring. DNA was extracted from autopsy spleen and bone marrow of 3B/M; from liver biopsies collected at 19 and 31 months and PBMCs obtained at 34 and 41 months after birth from 4B/M; and from liver, spleen, and bone marrow collected at autopsy of 5C/F performed at 22 months of age. The PBMCs harvested from 4B/M at 41 months after birth were extensively washed and the cell surface treated with DNase and trypsin before DNA isolation. DNA samples (2 or 5 μg) were digested with mung bean endonuclease and subjected to nested PCR with primers amplifying the WHV gap-spanning region. DNA samples from a WHsAg-positive chronic carrier were included as positive controls; water instead of DNA and a mock sample extracted in parallel with test samples were used as negative controls. Positive samples showed the expected size of the amplified nucleotide fragments indicated on the left.
    Figure Legend Snippet: Detection of WHV cccDNA in selected WHV DNA–reactive liver, PBMCs, and lymphoid tissue samples from 3B/M, 4B/M, and 5C/F offspring. DNA was extracted from autopsy spleen and bone marrow of 3B/M; from liver biopsies collected at 19 and 31 months and PBMCs obtained at 34 and 41 months after birth from 4B/M; and from liver, spleen, and bone marrow collected at autopsy of 5C/F performed at 22 months of age. The PBMCs harvested from 4B/M at 41 months after birth were extensively washed and the cell surface treated with DNase and trypsin before DNA isolation. DNA samples (2 or 5 μg) were digested with mung bean endonuclease and subjected to nested PCR with primers amplifying the WHV gap-spanning region. DNA samples from a WHsAg-positive chronic carrier were included as positive controls; water instead of DNA and a mock sample extracted in parallel with test samples were used as negative controls. Positive samples showed the expected size of the amplified nucleotide fragments indicated on the left.

    Techniques Used: DNA Extraction, Nested PCR, Amplification

    Effect of DNase digestion on WHV DNA–reactive particles circulating in an offspring born to a mother convalescent from viral hepatitis. Serum obtained at 32 months after birth from 4B/M offspring with WHV DNA expression in both liver and PBMCs; purified WHV virions; and Mnl I-digested recombinant WHV DNA were centrifuged through 15% sucrose over a 60% sucrose cushion, as described in Methods. Fifteen fractions collected from the bottom of each gradient were tested for WHV DNA by PCR, and those showing highest WHV DNA reactivity were pooled and were digested with DNase (D). Tested samples included pooled fractions 1–3 (bottom) for WHV virions, 1–5 (bottom) and 8–12 (top) for 4B/M serum, and 9–11 (top) for recombinant WHV DNA. As controls, samples of the same pooled fractions, but this time not treated with DNase (ND), were used. DNA extracted from each sample was tested for WHV S gene sequences by nested PCR and Southern blot hybridization.
    Figure Legend Snippet: Effect of DNase digestion on WHV DNA–reactive particles circulating in an offspring born to a mother convalescent from viral hepatitis. Serum obtained at 32 months after birth from 4B/M offspring with WHV DNA expression in both liver and PBMCs; purified WHV virions; and Mnl I-digested recombinant WHV DNA were centrifuged through 15% sucrose over a 60% sucrose cushion, as described in Methods. Fifteen fractions collected from the bottom of each gradient were tested for WHV DNA by PCR, and those showing highest WHV DNA reactivity were pooled and were digested with DNase (D). Tested samples included pooled fractions 1–3 (bottom) for WHV virions, 1–5 (bottom) and 8–12 (top) for 4B/M serum, and 9–11 (top) for recombinant WHV DNA. As controls, samples of the same pooled fractions, but this time not treated with DNase (ND), were used. DNA extracted from each sample was tested for WHV S gene sequences by nested PCR and Southern blot hybridization.

    Techniques Used: Expressing, Purification, Recombinant, Polymerase Chain Reaction, Nested PCR, Southern Blot, Hybridization

    Analysis of WHV DNA expression in liver, PBMCs, and lymphoid tissues collected from 3B/M offspring. WHV gene sequences were identified by nested PCR using C and X gene–specific primers, followed by Southern blot hybridization of the amplified products to recombinant WHV DNA. Five micrograms of DNA extracted from liver samples collected at 6 months of age and at autopsy performed at 15 months after birth, and 1 μg DNA from PBMCs collected at 14.5 months of age and from spleen, lymph node, bone marrow, and skeletal muscle obtained at autopsy, were used for direct PCR amplification. Positive samples showed the expected molecular size of the amplified virus C and X gene fragments indicated on the left.
    Figure Legend Snippet: Analysis of WHV DNA expression in liver, PBMCs, and lymphoid tissues collected from 3B/M offspring. WHV gene sequences were identified by nested PCR using C and X gene–specific primers, followed by Southern blot hybridization of the amplified products to recombinant WHV DNA. Five micrograms of DNA extracted from liver samples collected at 6 months of age and at autopsy performed at 15 months after birth, and 1 μg DNA from PBMCs collected at 14.5 months of age and from spleen, lymph node, bone marrow, and skeletal muscle obtained at autopsy, were used for direct PCR amplification. Positive samples showed the expected molecular size of the amplified virus C and X gene fragments indicated on the left.

    Techniques Used: Expressing, Nested PCR, Southern Blot, Hybridization, Amplification, Recombinant, Polymerase Chain Reaction

    WHV DNA expression in serum, liver, and lymphoid cells of 260/M woodchuck after inoculation with serum from liver WHV DNA–negative 3B/M offspring. Five micrograms of total DNA from liver biopsies collected at ∼2 months before (1) and 2 months after (2) inoculation and at autopsy (3), and 1 μg of DNA from autopsy PBMCs, isolated splenocytes, and bone marrow obtained at 3.5 months after inoculation or from 50 μL of autopsy serum, were tested for WHV DNA by nested PCR using WHV C gene–specific primers and hybridization to WHV DNA probe. DNA from serum of a WHsAg-positive chronic carrier was included as a positive control, and water instead of DNA and a mock sample extracted in parallel with test samples were used negative controls. Positive samples showed the expected 428-bp nucleotide fragment noted by arrowhead.
    Figure Legend Snippet: WHV DNA expression in serum, liver, and lymphoid cells of 260/M woodchuck after inoculation with serum from liver WHV DNA–negative 3B/M offspring. Five micrograms of total DNA from liver biopsies collected at ∼2 months before (1) and 2 months after (2) inoculation and at autopsy (3), and 1 μg of DNA from autopsy PBMCs, isolated splenocytes, and bone marrow obtained at 3.5 months after inoculation or from 50 μL of autopsy serum, were tested for WHV DNA by nested PCR using WHV C gene–specific primers and hybridization to WHV DNA probe. DNA from serum of a WHsAg-positive chronic carrier was included as a positive control, and water instead of DNA and a mock sample extracted in parallel with test samples were used negative controls. Positive samples showed the expected 428-bp nucleotide fragment noted by arrowhead.

    Techniques Used: Expressing, Isolation, Nested PCR, Hybridization, Positive Control

    Detection of WHV DNA in serial serum samples from 4B/M offspring. Sera collected between 2 and 36 months after birth were tested for WHV DNA by nested PCR using WHV C, S, and X gene–specific primers, and the amplified products were analyzed by Southern blot hybridization to recombinant WHV DNA probe. DNA isolated from serum of a WHsAg-positive chronic carrier and water were used as positive control and negative control, respectively. Positive samples show the expected sizes (bp) of the amplified nucleotide fragments noted by arrowheads.
    Figure Legend Snippet: Detection of WHV DNA in serial serum samples from 4B/M offspring. Sera collected between 2 and 36 months after birth were tested for WHV DNA by nested PCR using WHV C, S, and X gene–specific primers, and the amplified products were analyzed by Southern blot hybridization to recombinant WHV DNA probe. DNA isolated from serum of a WHsAg-positive chronic carrier and water were used as positive control and negative control, respectively. Positive samples show the expected sizes (bp) of the amplified nucleotide fragments noted by arrowheads.

    Techniques Used: Nested PCR, Amplification, Southern Blot, Hybridization, Recombinant, Isolation, Positive Control, Negative Control

    22) Product Images from "A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by 3-D tomography in the caveola-vesicle complexes (Sch?ffner's dots) of infected erythrocytes is a member of the PHIST family"

    Article Title: A 95 kDa protein of Plasmodium vivax and P. cynomolgi visualized by 3-D tomography in the caveola-vesicle complexes (Sch?ffner's dots) of infected erythrocytes is a member of the PHIST family

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2012.08060.x

    Protein structure and sequence identity of PcyPHIST/CVC-81 95 and its homologs. A. The schematic represents the PvPHIST/CVC-81 95 , PcyPHIST/CVC-81 95 , PkPHIST-105 and PfPHIST-147 proteins, showing their number of amino acids and main features as indicated.
    Figure Legend Snippet: Protein structure and sequence identity of PcyPHIST/CVC-81 95 and its homologs. A. The schematic represents the PvPHIST/CVC-81 95 , PcyPHIST/CVC-81 95 , PkPHIST-105 and PfPHIST-147 proteins, showing their number of amino acids and main features as indicated.

    Techniques Used: Sequencing

    PcyPHIST/CVC-81 95 is expressed in the ring, trophozoite and schizont stages of development.
    Figure Legend Snippet: PcyPHIST/CVC-81 95 is expressed in the ring, trophozoite and schizont stages of development.

    Techniques Used:

    A. P. cynomolgi phist/cvc-81 95 transfection experiments result in retrieval of episomes conferring resistance to pyrimethamine, but without integration in the genome. Results are shown for one of two transfection experiments. A. Schematic of the pcyphist/cvc-81
    Figure Legend Snippet: A. P. cynomolgi phist/cvc-81 95 transfection experiments result in retrieval of episomes conferring resistance to pyrimethamine, but without integration in the genome. Results are shown for one of two transfection experiments. A. Schematic of the pcyphist/cvc-81

    Techniques Used: Transfection

    23) Product Images from "Whole-exome sequencing reveals LRP5 mutations and canonical Wnt signaling associated with hepatic cystogenesis"

    Article Title: Whole-exome sequencing reveals LRP5 mutations and canonical Wnt signaling associated with hepatic cystogenesis

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

    doi: 10.1073/pnas.1309438111

    Whole-Exome Sequencing Identifies Pathogenic LRP5 Variant.
    Figure Legend Snippet: Whole-Exome Sequencing Identifies Pathogenic LRP5 Variant.

    Techniques Used: Sequencing, Variant Assay

    Whole-Exome Sequencing Identifies Pathogenic LRP5 Variant.
    Figure Legend Snippet: Whole-Exome Sequencing Identifies Pathogenic LRP5 Variant.

    Techniques Used: Sequencing, Variant Assay

    Identification of LRP5 variants p.R1188W in an extended Dutch PCLD-1 family ( A ) and three additional LRP5 variants in three unrelated PCLD families. Generations are denoted with Roman numerals, and individuals are numbered in a counterclockwise way. Squares
    Figure Legend Snippet: Identification of LRP5 variants p.R1188W in an extended Dutch PCLD-1 family ( A ) and three additional LRP5 variants in three unrelated PCLD families. Generations are denoted with Roman numerals, and individuals are numbered in a counterclockwise way. Squares

    Techniques Used:

    Functional and structural analyses of LRP5 variants in polycystic liver disease. ( A ) Immunohistochemistry of liver cyst tissue from proband III/18 of PCLD-1 family with LRP5 mutation c.3562C > T (p.R1188W). The cyst lining cholangiocytes present
    Figure Legend Snippet: Functional and structural analyses of LRP5 variants in polycystic liver disease. ( A ) Immunohistochemistry of liver cyst tissue from proband III/18 of PCLD-1 family with LRP5 mutation c.3562C > T (p.R1188W). The cyst lining cholangiocytes present

    Techniques Used: Functional Assay, Immunohistochemistry, Mutagenesis

    24) Product Images from "A Single Nucleotide Polymorphism in 3?-Untranslated Region Contributes to the Regulation of Toll-like Receptor 4 Translation *"

    Article Title: A Single Nucleotide Polymorphism in 3?-Untranslated Region Contributes to the Regulation of Toll-like Receptor 4 Translation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.338426

    Expression of TLR4 mRNA in PBMCs from subjects classified by rs11536889 genotype. PBMCs were isolated from the G/G, G/C, and C/C subjects, and total RNA was extracted. After reverse transcription, mRNA levels for TLR4 were determined by qRT-PCR using
    Figure Legend Snippet: Expression of TLR4 mRNA in PBMCs from subjects classified by rs11536889 genotype. PBMCs were isolated from the G/G, G/C, and C/C subjects, and total RNA was extracted. After reverse transcription, mRNA levels for TLR4 were determined by qRT-PCR using

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    25) Product Images from "Developmentally regulated expression and complex processing of barley pri-microRNAs"

    Article Title: Developmentally regulated expression and complex processing of barley pri-microRNAs

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-34

    Schematic representation of the MIR171e gene and its precursors. Detection of pri-, pre- and mature miR171e. ( A ) MIR171e gene structure. ( B ) pre-miRNA171e hairpin structure (ΔG=−59.1 kcal/mol) and its rice orthologue (ΔG=−58.9 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA171e structures (upper panel), green and yellow colors show alternatively retained transcript fragments as a consequence of alternative splicing events; RT-PCR detection of pri-miRNA171e expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA171e expression levels (upper graph) and its splice variants (I–IV) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR171e molecule, detection of pre-miRNA171e long (L) and short (S) intermediates, and mature miR171e using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .
    Figure Legend Snippet: Schematic representation of the MIR171e gene and its precursors. Detection of pri-, pre- and mature miR171e. ( A ) MIR171e gene structure. ( B ) pre-miRNA171e hairpin structure (ΔG=−59.1 kcal/mol) and its rice orthologue (ΔG=−58.9 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA171e structures (upper panel), green and yellow colors show alternatively retained transcript fragments as a consequence of alternative splicing events; RT-PCR detection of pri-miRNA171e expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA171e expression levels (upper graph) and its splice variants (I–IV) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR171e molecule, detection of pre-miRNA171e long (L) and short (S) intermediates, and mature miR171e using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR1120 gene and its precursor. Detection of pri-, pre- and mature miR1120. ( A ) MIR1120 gene structure; black squares in the gene and pri-miRNA1120 schemes show position of the ORF. ( B ) pre-miRNA1120 hairpin structure (ΔG=−42.3 kcal/mol) and its wheat orthologue (ΔG=−63.5 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA1120 structure and RT-PCR expression analysis in the five barley developmental stages studied. ( D ) Real-time PCR measurements of total pri-miRNA1120 expression levels; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR1120 molecule, and detection of pre-miRNA and mature miR1120 using Northern hybridization. U6 was used as a loading control. The level of pre-miRNAs and miRNA was calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Asterisk on agarose gel indicates unspecific product.
    Figure Legend Snippet: Schematic representation of the MIR1120 gene and its precursor. Detection of pri-, pre- and mature miR1120. ( A ) MIR1120 gene structure; black squares in the gene and pri-miRNA1120 schemes show position of the ORF. ( B ) pre-miRNA1120 hairpin structure (ΔG=−42.3 kcal/mol) and its wheat orthologue (ΔG=−63.5 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA1120 structure and RT-PCR expression analysis in the five barley developmental stages studied. ( D ) Real-time PCR measurements of total pri-miRNA1120 expression levels; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR1120 molecule, and detection of pre-miRNA and mature miR1120 using Northern hybridization. U6 was used as a loading control. The level of pre-miRNAs and miRNA was calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Asterisk on agarose gel indicates unspecific product.

    Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot, Agarose Gel Electrophoresis

    Schematic representation of the MIR1126 gene and its precursors. Detection of pri-, pre- and mature miR1126. ( A ) MIR1126 gene structure. ( B ) pre-miRNA1126 hairpin structure (ΔG=−78.4 kcal/mol) and its wheat orthologue (ΔG=−73.27 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–V) of the miR1126 transcript; dashed lines represents unamplified 5 ′ fragments of the noncoding RNA isoforms IV and V; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR expression analysis of splice isoforms (I–V) of the miR1126 transcript in all barley developmental stages studied. Half-open arrows on agarose gel indicate specific, identified products. ( E ) Real-time PCR measurements of total pri-miRNA1126 expression levels (upper graph) and pri-miR1126 fragments carrying the third intron (+IVS3) and after the third intron splicing (ΔIVS3) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( F ) Nucleotide sequence of the mature miR1126 molecule, and detection of pre-miRNA and mature miR1126 using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .
    Figure Legend Snippet: Schematic representation of the MIR1126 gene and its precursors. Detection of pri-, pre- and mature miR1126. ( A ) MIR1126 gene structure. ( B ) pre-miRNA1126 hairpin structure (ΔG=−78.4 kcal/mol) and its wheat orthologue (ΔG=−73.27 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–V) of the miR1126 transcript; dashed lines represents unamplified 5 ′ fragments of the noncoding RNA isoforms IV and V; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR expression analysis of splice isoforms (I–V) of the miR1126 transcript in all barley developmental stages studied. Half-open arrows on agarose gel indicate specific, identified products. ( E ) Real-time PCR measurements of total pri-miRNA1126 expression levels (upper graph) and pri-miR1126 fragments carrying the third intron (+IVS3) and after the third intron splicing (ΔIVS3) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( F ) Nucleotide sequence of the mature miR1126 molecule, and detection of pre-miRNA and mature miR1126 using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Techniques Used: Hybridization, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR159b gene and its precursors. Detection of pri- and mature miR159b. ( A ) MIR159b gene structure. ( B ) pre-miRNA159b hairpin structure (ΔG=−95 kcal/mol) and its rice orthologue (ΔG=−79.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 ( C ) pri-miRNA159b structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA159b expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR159b molecule, and detection of mature miR159b using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisks next to bands on agarose gel mark nonspecific products.
    Figure Legend Snippet: Schematic representation of the MIR159b gene and its precursors. Detection of pri- and mature miR159b. ( A ) MIR159b gene structure. ( B ) pre-miRNA159b hairpin structure (ΔG=−95 kcal/mol) and its rice orthologue (ΔG=−79.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 ( C ) pri-miRNA159b structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA159b expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR159b molecule, and detection of mature miR159b using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisks next to bands on agarose gel mark nonspecific products.

    Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot, Agarose Gel Electrophoresis

    Schematic representation of the MIR166n gene and its precursors. Detection of pri-, pre- and mature miR166n. ( A ) MIR166n gene structure. ( B ) pre-miRNA166n hairpin structure (ΔG=−61 kcal/mol) and its rice orthologue (ΔG=−52.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA166n structures (upper panel); RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA166n expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR166n molecule, and detection of pre-miRNA166n long (L) and short (S) intermediates, and mature miR166n using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .
    Figure Legend Snippet: Schematic representation of the MIR166n gene and its precursors. Detection of pri-, pre- and mature miR166n. ( A ) MIR166n gene structure. ( B ) pre-miRNA166n hairpin structure (ΔG=−61 kcal/mol) and its rice orthologue (ΔG=−52.3 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA166n structures (upper panel); RT-PCR analysis of their expression in five barley developmental stages studied (lower panel). ( D ) Real-time PCR measurements of total pri-miRNA166n expression level (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miR166n molecule, and detection of pre-miRNA166n long (L) and short (S) intermediates, and mature miR166n using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 .

    Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR168a-5p/168-3p gene and its precursors. Detection of pri-, pre-, and mature miR168-5p and miR168a-3p. ( A ) MIR168a-5p/168-3p gene structure. ( B ) pre-miRNA168a-5p/168-3p hairpin structure (ΔG=−60.7 kcal/mol) and its rice orthologue (ΔG=−52.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA168a-5p/168-3p structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of pri-miRNA miRNA168a-5p/168-3p expression levels (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequences of the mature miR168a-5p and miR168a-3p molecules, and Northern detection of pre-miRNA168a-5p/168-3p long (L) and short (S) intermediates, mature miR168-5p and miR168a-3p. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisk next to band on agarose gel marks nonspecific product.
    Figure Legend Snippet: Schematic representation of the MIR168a-5p/168-3p gene and its precursors. Detection of pri-, pre-, and mature miR168-5p and miR168a-3p. ( A ) MIR168a-5p/168-3p gene structure. ( B ) pre-miRNA168a-5p/168-3p hairpin structure (ΔG=−60.7 kcal/mol) and its rice orthologue (ΔG=−52.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) pri-miRNA168a-5p/168-3p structures (upper panel) and RT-PCR analysis of their expression in five barley developmental stages (lower panel). ( D ) Real-time PCR measurements of pri-miRNA miRNA168a-5p/168-3p expression levels (upper graph) and its spliced (ΔIVS) and unspliced variants (+IVS) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( E ) Nucleotide sequences of the mature miR168a-5p and miR168a-3p molecules, and Northern detection of pre-miRNA168a-5p/168-3p long (L) and short (S) intermediates, mature miR168-5p and miR168a-3p. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 ; asterisk next to band on agarose gel marks nonspecific product.

    Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Northern Blot, Agarose Gel Electrophoresis

    Schematic representation of the MIR156g gene and its precursors. Detection of pri-, pre- and mature miR156g. ( A ) MIR156g gene structure; thin black vertical bars within exons show additional splice sites identified during pri-miRNA156g analyses; dotted-vertical lines within the last exon together with pA symbols denote polyadenylation sites. ( B ) pre-miRNA156g hairpin structure (ΔG=−65.85 kcal/mol) and its rice orthologue (ΔG=−61.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–VIII) of the miR156g transcript; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR analysis of first intron retention throughout barley plant life stages. ( E–F ) pri-miRNA156g RT-PCR expression analysis in five barley developmental stages. Arrows on agarose gel indicate splice isoforms II, III and V. ( G ) Real-time PCR measurements of total pri-miRNA156g expression levels (upper graph) and pri-miR156g fragments carrying the first intron (+IVS1) and after the first intron splicing (ΔIVS1) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( H ) Nucleotide sequence of the mature miR156g molecule, and detection of pre-miRNA and mature miR156g using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Additional colors depict alternatively spliced exons in the pri-miRNA.
    Figure Legend Snippet: Schematic representation of the MIR156g gene and its precursors. Detection of pri-, pre- and mature miR156g. ( A ) MIR156g gene structure; thin black vertical bars within exons show additional splice sites identified during pri-miRNA156g analyses; dotted-vertical lines within the last exon together with pA symbols denote polyadenylation sites. ( B ) pre-miRNA156g hairpin structure (ΔG=−65.85 kcal/mol) and its rice orthologue (ΔG=−61.2 kcal/mol); blue and red lines indicate hybridization regions as described in Figure 1 . ( C ) Structures of splice isoforms (I–VIII) of the miR156g transcript; …polyA indicates a putative polyA site in splice isoforms as the determination of an accurate polyA site for PCR products is not possible. ( D ) RT-PCR analysis of first intron retention throughout barley plant life stages. ( E–F ) pri-miRNA156g RT-PCR expression analysis in five barley developmental stages. Arrows on agarose gel indicate splice isoforms II, III and V. ( G ) Real-time PCR measurements of total pri-miRNA156g expression levels (upper graph) and pri-miR156g fragments carrying the first intron (+IVS1) and after the first intron splicing (ΔIVS1) (lower graph); bars on the charts represent standard deviation. Values are shown as the mean ±SD (n=3) from three independent experiments. ( H ) Nucleotide sequence of the mature miR156g molecule, and detection of pre-miRNA and mature miR156g using Northern hybridization. U6 was used as a loading control. The levels of pre-miRNAs and miRNA were calculated as described in Figure 1 . Colors, abbreviations, and symbols as in Figure 1 . Additional colors depict alternatively spliced exons in the pri-miRNA.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    Schematic representation of the MIR397b-3p gene and its precursors. Detection of pri-, pre- and mature miR397b-3p. ( A ) MIR397b-3p gene structure; left arrow indicates putative transcription start site; arrow marked as pA depicts precursor polyadenylation site. ( B ) pre-miRNA397b-3p hairpin structure (ΔG=−70.8 kcal/mol) and its rice orthologue (ΔG=−51.2 kcal/mol); the blue line indicates the region of the pre-miRNA from which the hybridization probe for precursor detection was designed, while the red line highlights the probe for detection of the mature miRNA. ( C ) Structure of pri-miRNA397b-3p (upper panel); RT-PCR analysis of its expression in five barley developmental stages (lower panel); primer positions are marked by black triangles on the pri-miRNA graph. ( D ) Real-time PCR measurements of pri-miRNA397b-3p expression level; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miRNA397b-3p molecule; detection of pre-miRNA (left upper panel), mature miR397b-3p (left middle panel), and miR397b-5p (right panel) using Northern hybridization. U6 was used as a loading control. The level of pre-miRNA and miRNA in 1-week-old plants was arbitrarily assumed to be ‘1’, and the levels of pre-miRNA and miRNA were quantified relative to this at all other developmental stages. The miRNA is marked in red, the miRNA* in blue; 1w: one-week-old seedlings, 2w: two-week-old seedlings, 3w: three-week-old plants, 6w: six-week-old plants, 68d: 68-day-old plants, gDNA: genomic DNA; M - GeneRuler 100 bp Plus or 1kb Plus DNA Ladders.
    Figure Legend Snippet: Schematic representation of the MIR397b-3p gene and its precursors. Detection of pri-, pre- and mature miR397b-3p. ( A ) MIR397b-3p gene structure; left arrow indicates putative transcription start site; arrow marked as pA depicts precursor polyadenylation site. ( B ) pre-miRNA397b-3p hairpin structure (ΔG=−70.8 kcal/mol) and its rice orthologue (ΔG=−51.2 kcal/mol); the blue line indicates the region of the pre-miRNA from which the hybridization probe for precursor detection was designed, while the red line highlights the probe for detection of the mature miRNA. ( C ) Structure of pri-miRNA397b-3p (upper panel); RT-PCR analysis of its expression in five barley developmental stages (lower panel); primer positions are marked by black triangles on the pri-miRNA graph. ( D ) Real-time PCR measurements of pri-miRNA397b-3p expression level; bars on a chart represent standard deviation. Values are shown as the mean ± SD (n=3) from three independent experiments. ( E ) Nucleotide sequence of the mature miRNA397b-3p molecule; detection of pre-miRNA (left upper panel), mature miR397b-3p (left middle panel), and miR397b-5p (right panel) using Northern hybridization. U6 was used as a loading control. The level of pre-miRNA and miRNA in 1-week-old plants was arbitrarily assumed to be ‘1’, and the levels of pre-miRNA and miRNA were quantified relative to this at all other developmental stages. The miRNA is marked in red, the miRNA* in blue; 1w: one-week-old seedlings, 2w: two-week-old seedlings, 3w: three-week-old plants, 6w: six-week-old plants, 68d: 68-day-old plants, gDNA: genomic DNA; M - GeneRuler 100 bp Plus or 1kb Plus DNA Ladders.

    Techniques Used: Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Standard Deviation, Sequencing, Northern Blot

    26) Product Images from "Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies"

    Article Title: Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm016

    Biochemical characterization of hMex-3 proteins. ( A and B ) BOSC cells were transiently transfected with vectors expressing myc-tagged forms of hMex-3A, -3B and -3C. Western blot analysis was performed with anti-myc antibody. In (B) treatment of protein extracts with (+) or without (−) λ-Phosphatase. ( C ) Kinase assay. hMex-3A and -3B proteins or a control protein (Bpag1) expressed in BOSC cells were immunoprecipitated with the anti-myc antibody and incubated with kinase buffer and [γ 32 P] ATP. Labeled proteins were revealed by autoradiography. ( D ) RNA homopolymer binding assay. Proteins from indicated expression vectors were in vitro translated in the presence of [ 35 S] methionine ( top ). Binding to agarose beads coupled to poly(A) ( bottom ) RNA homopolymers is shown for in vitro translated proteins. As a negative control, a fragment of P62-sequestosome protein was incubated with RNA homopolymers in the same conditions. One-tenth of the initial translation reactions and all the bound proteins were analysed by SDS-PAGE and autoradiography. ( E ) In vivo hMex-3 binding to mRNA. BOSC cells were transiently transfected with vectors expressing myc-tagged proteins, as indicated. RT-PCR amplification was performed on total RNA extracted from those cells ( top left ). Western blot analysis performed with anti-myc antibody ( bottom left ). RT-PCR amplification performed on total RNA extracted from sepharose-protein A beads after immunoprecipitation by an anti-myc antibody ( right ).
    Figure Legend Snippet: Biochemical characterization of hMex-3 proteins. ( A and B ) BOSC cells were transiently transfected with vectors expressing myc-tagged forms of hMex-3A, -3B and -3C. Western blot analysis was performed with anti-myc antibody. In (B) treatment of protein extracts with (+) or without (−) λ-Phosphatase. ( C ) Kinase assay. hMex-3A and -3B proteins or a control protein (Bpag1) expressed in BOSC cells were immunoprecipitated with the anti-myc antibody and incubated with kinase buffer and [γ 32 P] ATP. Labeled proteins were revealed by autoradiography. ( D ) RNA homopolymer binding assay. Proteins from indicated expression vectors were in vitro translated in the presence of [ 35 S] methionine ( top ). Binding to agarose beads coupled to poly(A) ( bottom ) RNA homopolymers is shown for in vitro translated proteins. As a negative control, a fragment of P62-sequestosome protein was incubated with RNA homopolymers in the same conditions. One-tenth of the initial translation reactions and all the bound proteins were analysed by SDS-PAGE and autoradiography. ( E ) In vivo hMex-3 binding to mRNA. BOSC cells were transiently transfected with vectors expressing myc-tagged proteins, as indicated. RT-PCR amplification was performed on total RNA extracted from those cells ( top left ). Western blot analysis performed with anti-myc antibody ( bottom left ). RT-PCR amplification performed on total RNA extracted from sepharose-protein A beads after immunoprecipitation by an anti-myc antibody ( right ).

    Techniques Used: Transfection, Expressing, Western Blot, Kinase Assay, Immunoprecipitation, Incubation, Labeling, Autoradiography, Binding Assay, In Vitro, Negative Control, SDS Page, In Vivo, Reverse Transcription Polymerase Chain Reaction, Amplification

    Expression profile of hMex -3 genes and hMex-3B protein. ( A ) Human Mex -3 gene expression levels ( panels 1–4 ) were examined by RT-PCR with specific internal primers and were compared with expression level of ubiquitously expressed GAPDH gene ( panel 5 ). RNA were extracted from 7 human cell lines ( left ) and from 20 human tissues (Multiple Tissue Total RNA panel, BD Biosciences) ( right ). ( B ) Human colon sections (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ antibody ( panels 1 and 2 ), anti-hMex-3Bβ antibody + hMex-3B peptide, as control ( panel 3 ). ( C ) Serial sections of human Meckel's diverticulum (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ ( top left ) and anti-MUC2 ( bottom left ), anti-hMex-3Bβ ( top middle ) and anti-Chromogranin-A ( bottom middle ), anti-hMex-3Bβ ( top right ) and anti-Lysozyme ( bottom right ).
    Figure Legend Snippet: Expression profile of hMex -3 genes and hMex-3B protein. ( A ) Human Mex -3 gene expression levels ( panels 1–4 ) were examined by RT-PCR with specific internal primers and were compared with expression level of ubiquitously expressed GAPDH gene ( panel 5 ). RNA were extracted from 7 human cell lines ( left ) and from 20 human tissues (Multiple Tissue Total RNA panel, BD Biosciences) ( right ). ( B ) Human colon sections (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ antibody ( panels 1 and 2 ), anti-hMex-3Bβ antibody + hMex-3B peptide, as control ( panel 3 ). ( C ) Serial sections of human Meckel's diverticulum (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ ( top left ) and anti-MUC2 ( bottom left ), anti-hMex-3Bβ ( top middle ) and anti-Chromogranin-A ( bottom middle ), anti-hMex-3Bβ ( top right ) and anti-Lysozyme ( bottom right ).

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

    27) Product Images from "The involvement of replication in single stranded oligonucleotide-mediated gene repair"

    Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl852

    Chain-terminating ddC residue prevents replicative extension in vitro . Primers containing 6 phosphothioate linkages at each terminus (PT SSO) or 6 phosphothioate linkages at each terminus and a 3′-dideoxycytidine residue (PT+ddC SSO) were used to amplify a 196 bp fragment using pGKfrtmCM(−) as template and mCM(+)DT2 as the reverse primer, in a standard PCR reaction (see Table 6 in Supplementary material for primer sequences). PCRs were performed with the modified SSOs present at three different concentrations (1, 10 or 100 ng per reaction). Results show that the chain-terminating ddC nucleotide on the PT+ddC SSO is sufficient to prevent replicative extension by a DNA polymerase endowed with proofreading activity.
    Figure Legend Snippet: Chain-terminating ddC residue prevents replicative extension in vitro . Primers containing 6 phosphothioate linkages at each terminus (PT SSO) or 6 phosphothioate linkages at each terminus and a 3′-dideoxycytidine residue (PT+ddC SSO) were used to amplify a 196 bp fragment using pGKfrtmCM(−) as template and mCM(+)DT2 as the reverse primer, in a standard PCR reaction (see Table 6 in Supplementary material for primer sequences). PCRs were performed with the modified SSOs present at three different concentrations (1, 10 or 100 ng per reaction). Results show that the chain-terminating ddC nucleotide on the PT+ddC SSO is sufficient to prevent replicative extension by a DNA polymerase endowed with proofreading activity.

    Techniques Used: In Vitro, Polymerase Chain Reaction, Modification, Activity Assay

    Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.
    Figure Legend Snippet: Verification of SSO incorporation into its homologous DNA target ( A ) A schematic illustration of the experimental procedure. Biotinylated recombination products were purified using magnetic streptavidin beads. The presence of (corrected) pmKan was confirmed by the detection of a 496 bp PCR product. ( B ) pmKan and ddH 2 O were used as templates for the negative and positive PCR controls (lanes 2 and 3 respectively). DY380/pmKan cells were incubated at 42°C for 15 min to induce λ-Red protein expression prior to electroporation with biotinylated-SSO (lane 6) or unmodified SSO (lane 4). As a control, DY380/pmKan cells that had been incubated at 32°C for 15 min (i.e. no λ-Red induction) were similarly electroporated with biotinylated-SSO (lane 5). Plasmid DNA were extracted from the electroporated cells after a 15 min recovery period. Three independent experiments were performed; a representative experiment is shown.

    Techniques Used: Purification, Polymerase Chain Reaction, Incubation, Expressing, Electroporation, Plasmid Preparation

    28) Product Images from "A multiple-site-specific heteroduplex tracking assay as a tool for the study of viral population dynamics"

    Article Title: A multiple-site-specific heteroduplex tracking assay as a tool for the study of viral population dynamics

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

    doi:

    Reproducibility and sensitivity of the pro MSS HTA. ( A ) The MSS HTA reflects the populations in the PCR product accurately and reproducibly at high template numbers. PCR products of two pro genes were mixed at known ratios; the mixtures were then diluted over a 100-fold range and annealed to the MSS HTA probe 6.1. The abundance of one of the products was determined by using the MSS HTA and compared with the known abundance. Error bars represent standard deviations from 5–9 experiments. ( B ) Amplification of pro gene mixtures. Mixtures of pro ) T12S, K43R, M46I, I54V, Q61H, L63P, V82F.
    Figure Legend Snippet: Reproducibility and sensitivity of the pro MSS HTA. ( A ) The MSS HTA reflects the populations in the PCR product accurately and reproducibly at high template numbers. PCR products of two pro genes were mixed at known ratios; the mixtures were then diluted over a 100-fold range and annealed to the MSS HTA probe 6.1. The abundance of one of the products was determined by using the MSS HTA and compared with the known abundance. Error bars represent standard deviations from 5–9 experiments. ( B ) Amplification of pro gene mixtures. Mixtures of pro ) T12S, K43R, M46I, I54V, Q61H, L63P, V82F.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Cross-sectional study of viral pro populations and correlation of MSS HTA mobility shifts with reduced drug susceptibility. ( A ) The MSS HTA analysis of RT-PCR products from 21 patient plasma samples with different treatment histories. Differences in the bulk sequence of the RT-PCR products from HIV-1 clade B consensus are shown above the gel. Two amino acids indicate a mixed population. Note that positions that were not resistance-associated were omitted. Note also that each population contains at least one of the targeted mutations of the MSS HTA probe (highlighted in gray). In 11/21 cases, multiple populations differing at or near the targeted positions were found by HTA, whereas population-based sequencing identified mixed populations in only 4 subjects. hd, heteroduplex; dsP, double-stranded probe. ( B ) Mobility ( k ) of the most prominent MSS HTA band of each subject correlated with the average reduction of susceptibility to ritonavir, saquinavir, and indinavir ( r a ) of the complete virus population compared with molecular clone NL4–3 (○). The labels indicate the number of targeted mutations seen in the bulk sequence. For comparison, the mobility of bands corresponding to viral populations from seven protease inhibitor-naïve patients (⋄) and the mobility of molecular clones NL4–3 and Hxb-2r (♦) are shown. Not all of these are visible on the plot, because some mobilities are identical.
    Figure Legend Snippet: Cross-sectional study of viral pro populations and correlation of MSS HTA mobility shifts with reduced drug susceptibility. ( A ) The MSS HTA analysis of RT-PCR products from 21 patient plasma samples with different treatment histories. Differences in the bulk sequence of the RT-PCR products from HIV-1 clade B consensus are shown above the gel. Two amino acids indicate a mixed population. Note that positions that were not resistance-associated were omitted. Note also that each population contains at least one of the targeted mutations of the MSS HTA probe (highlighted in gray). In 11/21 cases, multiple populations differing at or near the targeted positions were found by HTA, whereas population-based sequencing identified mixed populations in only 4 subjects. hd, heteroduplex; dsP, double-stranded probe. ( B ) Mobility ( k ) of the most prominent MSS HTA band of each subject correlated with the average reduction of susceptibility to ritonavir, saquinavir, and indinavir ( r a ) of the complete virus population compared with molecular clone NL4–3 (○). The labels indicate the number of targeted mutations seen in the bulk sequence. For comparison, the mobility of bands corresponding to viral populations from seven protease inhibitor-naïve patients (⋄) and the mobility of molecular clones NL4–3 and Hxb-2r (♦) are shown. Not all of these are visible on the plot, because some mobilities are identical.

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

    Development of an RT MSS HTA. ( A ) are shown above. ( B ) Mobility of the radioactively labeled probe when annealed to three PCR products containing resistance mutations in comparison with the mobility of wild type (wt). In addition, plasma RNA of Patient 1029 who started 3TC therapy at day 215 was subjected to an MSS HTA analysis shown on the same gel. hd, heteroduplex; dsP, double-stranded probe.
    Figure Legend Snippet: Development of an RT MSS HTA. ( A ) are shown above. ( B ) Mobility of the radioactively labeled probe when annealed to three PCR products containing resistance mutations in comparison with the mobility of wild type (wt). In addition, plasma RNA of Patient 1029 who started 3TC therapy at day 215 was subjected to an MSS HTA analysis shown on the same gel. hd, heteroduplex; dsP, double-stranded probe.

    Techniques Used: Labeling, Polymerase Chain Reaction

    Characteristics of the pro MSS HTA probe 6.1. ( A ). Resistance-relevant changes are in close proximity to probe wild-type mismatches. ( B ) Mobility of the radioactively labeled probe annealed to PCR products of pro genes with point mutations. Only the heteroduplexes (hd) and the probe that annealed to its fully complementary strand (double-stranded probe, dsP) are shown. Lanes: 1, wild type; 2, M46I; 3, G48V; 4, I54T; 5, L63P; 6, V82T; 7, V82A; 8, I84V; 9, L90M; 10, G48V/V82T; and 11, G48V/L90M. The mobility ( k ) of each hd relative to the dsP is indicated above all lanes. Note that wild type and L63P, a nontargeted mutation, have identical mobilities, whereas all of the targeted mutations display lower mobilities. The mobilities of the hds are calculated relative to the dsP to control for differences in the gel or in the electric field between lanes and gels.
    Figure Legend Snippet: Characteristics of the pro MSS HTA probe 6.1. ( A ). Resistance-relevant changes are in close proximity to probe wild-type mismatches. ( B ) Mobility of the radioactively labeled probe annealed to PCR products of pro genes with point mutations. Only the heteroduplexes (hd) and the probe that annealed to its fully complementary strand (double-stranded probe, dsP) are shown. Lanes: 1, wild type; 2, M46I; 3, G48V; 4, I54T; 5, L63P; 6, V82T; 7, V82A; 8, I84V; 9, L90M; 10, G48V/V82T; and 11, G48V/L90M. The mobility ( k ) of each hd relative to the dsP is indicated above all lanes. Note that wild type and L63P, a nontargeted mutation, have identical mobilities, whereas all of the targeted mutations display lower mobilities. The mobilities of the hds are calculated relative to the dsP to control for differences in the gel or in the electric field between lanes and gels.

    Techniques Used: Labeling, Polymerase Chain Reaction, Mutagenesis

    29) Product Images from "Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis"

    Article Title: Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.6.3234-3248.2002

    RT-PCR analysis of IDO mRNA expression in dTHP-1 cells and MdM stimulated for 24 h with C. trachomatis -infected HeLa cell supernatants and in dTHP-1 cells cocultivated with infected HeLa cells for 24 and 48 h. The cDNAs were amplified with IDO (324 bp, top panel) and GAPDH (306 bp, bottom panel) primers for 35 and 22 PCR cycles, respectively. A total of 10 μl of PCR products was loaded on a 2% agarose gel as follows. dTHP-1 cells and MdM were incubated with supernatants from HeLa cells left uninfected (lanes 1 and 6, respectively), infected with serovar E (lanes 2 and 7, respectively) or serovar L2 (lanes 3 and 8, respectively), treated with RPMI alone (lanes 4 and 9, respectively) or E. coli LPS alone (lanes 5 and 10, respectively), or dTHP-1 cells were incubated in coculture for 24 and 48 h with uninfected HeLa cells (lanes 11 and 14, respectively), with serovar E-infected HeLa cells (lanes 12 and 15, respectively), or with serovar L2-infected HeLa cells (lanes 13 and 16, respectively). A negative control for amplification (lane 17) wherein DNA was omitted and a positive control (lane 18) consisting of cDNA from HeLa cells exposed to rhIFN-γ (10 ng/ml for 12 h) were also included in the analysis.
    Figure Legend Snippet: RT-PCR analysis of IDO mRNA expression in dTHP-1 cells and MdM stimulated for 24 h with C. trachomatis -infected HeLa cell supernatants and in dTHP-1 cells cocultivated with infected HeLa cells for 24 and 48 h. The cDNAs were amplified with IDO (324 bp, top panel) and GAPDH (306 bp, bottom panel) primers for 35 and 22 PCR cycles, respectively. A total of 10 μl of PCR products was loaded on a 2% agarose gel as follows. dTHP-1 cells and MdM were incubated with supernatants from HeLa cells left uninfected (lanes 1 and 6, respectively), infected with serovar E (lanes 2 and 7, respectively) or serovar L2 (lanes 3 and 8, respectively), treated with RPMI alone (lanes 4 and 9, respectively) or E. coli LPS alone (lanes 5 and 10, respectively), or dTHP-1 cells were incubated in coculture for 24 and 48 h with uninfected HeLa cells (lanes 11 and 14, respectively), with serovar E-infected HeLa cells (lanes 12 and 15, respectively), or with serovar L2-infected HeLa cells (lanes 13 and 16, respectively). A negative control for amplification (lane 17) wherein DNA was omitted and a positive control (lane 18) consisting of cDNA from HeLa cells exposed to rhIFN-γ (10 ng/ml for 12 h) were also included in the analysis.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Incubation, Negative Control, Positive Control

    30) Product Images from "Sequence Variations of Full-Length Hepatitis B Virus Genomes in Chinese Patients with HBsAg-Negative Hepatitis B Infection"

    Article Title: Sequence Variations of Full-Length Hepatitis B Virus Genomes in Chinese Patients with HBsAg-Negative Hepatitis B Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099028

    Rolling circle amplification (RCA) of full-length HBV genome. The number of initial HBV DNA template present in each RCA reaction is shown at the top. M, molecular weight marker; H2O, negative control. (A) The high-molecular-weight raw RCA products containing multiple copies of the initial HBV template. (B) The full-length HBV genomes recovered by restriction enzyme, Spe I digestion. (C) Full-length HBV genome amplified by using the RCA products as PCR template.
    Figure Legend Snippet: Rolling circle amplification (RCA) of full-length HBV genome. The number of initial HBV DNA template present in each RCA reaction is shown at the top. M, molecular weight marker; H2O, negative control. (A) The high-molecular-weight raw RCA products containing multiple copies of the initial HBV template. (B) The full-length HBV genomes recovered by restriction enzyme, Spe I digestion. (C) Full-length HBV genome amplified by using the RCA products as PCR template.

    Techniques Used: Amplification, Molecular Weight, Marker, Negative Control, Polymerase Chain Reaction

    31) Product Images from "Pericytoma with t(7;12) and ACTB-GLI1 Fusion Arising in Bone"

    Article Title: Pericytoma with t(7;12) and ACTB-GLI1 Fusion Arising in Bone

    Journal: Human pathology

    doi: 10.1016/j.humpath.2012.01.019

    A. Schematic illustration (top) and partial G-banded karyotype (bottom) illustrating the 7;12 translocation. B. RT-PCR detected ACTB-GLI1 fusion transcripts using ACTB 61F-868R and ACTB 18F-1246R primers, which amplified DNA products of 700 bp (lane 1) and 1119 bp (lane 2) respectively. M, 1 kb DNA ladder.
    Figure Legend Snippet: A. Schematic illustration (top) and partial G-banded karyotype (bottom) illustrating the 7;12 translocation. B. RT-PCR detected ACTB-GLI1 fusion transcripts using ACTB 61F-868R and ACTB 18F-1246R primers, which amplified DNA products of 700 bp (lane 1) and 1119 bp (lane 2) respectively. M, 1 kb DNA ladder.

    Techniques Used: Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

    32) Product Images from "Persistent Helicobacter pullorum colonization in C57BL/6NTac mice: a new mouse model for an emerging zoonosis"

    Article Title: Persistent Helicobacter pullorum colonization in C57BL/6NTac mice: a new mouse model for an emerging zoonosis

    Journal: Journal of Medical Microbiology

    doi: 10.1099/jmm.0.040055-0

    PCR amplification of a 148 bp product using H. pullorum -specific cdtB primers. Lanes: M, 1 kb Plus DNA ladder (Invitrogen); 1–3 and 10, H. pullorum -negative caecal cultures; 4–9 and 11, bacterial DNA of H. pullorum caecal isolates; 12,
    Figure Legend Snippet: PCR amplification of a 148 bp product using H. pullorum -specific cdtB primers. Lanes: M, 1 kb Plus DNA ladder (Invitrogen); 1–3 and 10, H. pullorum -negative caecal cultures; 4–9 and 11, bacterial DNA of H. pullorum caecal isolates; 12,

    Techniques Used: Polymerase Chain Reaction, Amplification

    33) Product Images from "Splicing Factor 1 Modulates Dietary Restriction and TORC1 Pathway Longevity in C. elegans"

    Article Title: Splicing Factor 1 Modulates Dietary Restriction and TORC1 Pathway Longevity in C. elegans

    Journal: Nature

    doi: 10.1038/nature20789

    RT-PCR validation of alternative splicing events in ageing and with sfa-1 knockdown a, Sequencing reads coverage for tos-1 b, Age-associated isoform ratio change of a target of SFA-1, target of splicing ( tos-1 ) in WT worms at day 3 and 15 of adulthood ± sfa-1 RNAi by RT-PCR (biological replicates 3 and 4 shown). c, Sequencing read coverage map for ret-1 shows increased exon 5 skipping with age and with sfa-1 RNAi. d, Endogenous ret-1 exon 5 splicing pattern with age and sfa-1 RNAi in WT and DR worms by RT-PCR (day 3 vs. 15, 2 biological replicates shown). e, Sequencing tracks for lipl-7 pre-mRNA. f, Monitoring of intron retention between exons 4 and 5 at day 15 vs. day 3 of adulthood in WT and DR worms, +/− sfa-1 RNAi. g, Sequencing reads tracks for slo-2 pre-mRNA. h, slo-2 alternative exon skipping in day 3 and day 15 old WT and DR worms, +/− sfa-1 RNAi. i, Sequencing reads tracks for lea-1 pre-mRNA j , Alternative exon skipping in lea-1 with age and sfa-1 knockdown in WT and DR animals. Sequencing reads tracks generated by Splicing Java Coverage Viewer as part of SAJR 29 ; height of red lines represent RNA coverage of splice junctions, dark gray boxes represent exonic sequence, light gray boxes are alternative exon sequence.
    Figure Legend Snippet: RT-PCR validation of alternative splicing events in ageing and with sfa-1 knockdown a, Sequencing reads coverage for tos-1 b, Age-associated isoform ratio change of a target of SFA-1, target of splicing ( tos-1 ) in WT worms at day 3 and 15 of adulthood ± sfa-1 RNAi by RT-PCR (biological replicates 3 and 4 shown). c, Sequencing read coverage map for ret-1 shows increased exon 5 skipping with age and with sfa-1 RNAi. d, Endogenous ret-1 exon 5 splicing pattern with age and sfa-1 RNAi in WT and DR worms by RT-PCR (day 3 vs. 15, 2 biological replicates shown). e, Sequencing tracks for lipl-7 pre-mRNA. f, Monitoring of intron retention between exons 4 and 5 at day 15 vs. day 3 of adulthood in WT and DR worms, +/− sfa-1 RNAi. g, Sequencing reads tracks for slo-2 pre-mRNA. h, slo-2 alternative exon skipping in day 3 and day 15 old WT and DR worms, +/− sfa-1 RNAi. i, Sequencing reads tracks for lea-1 pre-mRNA j , Alternative exon skipping in lea-1 with age and sfa-1 knockdown in WT and DR animals. Sequencing reads tracks generated by Splicing Java Coverage Viewer as part of SAJR 29 ; height of red lines represent RNA coverage of splice junctions, dark gray boxes represent exonic sequence, light gray boxes are alternative exon sequence.

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

    34) Product Images from "Type I Interferon Response Is Delayed in Human Astrovirus Infections"

    Article Title: Type I Interferon Response Is Delayed in Human Astrovirus Infections

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123087

    Induction of an IFN response is delayed during HAstV infection. (A) Temporal analysis of induction of IFN-β and ISG56 mRNA expression by in CaCo-2 cells infected with HAstV at a MOI of 1. Mock-infected cells, cells treated for 24 h with exogenous IFN at 1,000 U/ml, and polyI:C-transfected cells were used as controls. (B) HAstV growth curve on CaCo-2 cells at 2 different MOIs. Total HAstV RNA was measured by qRT-PCR at the indicated times post-infection. Data represent mean values of duplicate wells and error bars represent the standard error of the mean (SEM).
    Figure Legend Snippet: Induction of an IFN response is delayed during HAstV infection. (A) Temporal analysis of induction of IFN-β and ISG56 mRNA expression by in CaCo-2 cells infected with HAstV at a MOI of 1. Mock-infected cells, cells treated for 24 h with exogenous IFN at 1,000 U/ml, and polyI:C-transfected cells were used as controls. (B) HAstV growth curve on CaCo-2 cells at 2 different MOIs. Total HAstV RNA was measured by qRT-PCR at the indicated times post-infection. Data represent mean values of duplicate wells and error bars represent the standard error of the mean (SEM).

    Techniques Used: Infection, Expressing, Transfection, Quantitative RT-PCR

    35) Product Images from "Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis"

    Article Title: Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.6.3234-3248.2002

    RT-PCR analysis of IDO mRNA expression in dTHP-1 cells and MdM stimulated for 24 h with C. trachomatis -infected HeLa cell supernatants and in dTHP-1 cells cocultivated with infected HeLa cells for 24 and 48 h. The cDNAs were amplified with IDO (324 bp, top panel) and GAPDH (306 bp, bottom panel) primers for 35 and 22 PCR cycles, respectively. A total of 10 μl of PCR products was loaded on a 2% agarose gel as follows. dTHP-1 cells and MdM were incubated with supernatants from HeLa cells left uninfected (lanes 1 and 6, respectively), infected with serovar E (lanes 2 and 7, respectively) or serovar L2 (lanes 3 and 8, respectively), treated with RPMI alone (lanes 4 and 9, respectively) or E. coli LPS alone (lanes 5 and 10, respectively), or dTHP-1 cells were incubated in coculture for 24 and 48 h with uninfected HeLa cells (lanes 11 and 14, respectively), with serovar E-infected HeLa cells (lanes 12 and 15, respectively), or with serovar L2-infected HeLa cells (lanes 13 and 16, respectively). A negative control for amplification (lane 17) wherein DNA was omitted and a positive control (lane 18) consisting of cDNA from HeLa cells exposed to rhIFN-γ (10 ng/ml for 12 h) were also included in the analysis.
    Figure Legend Snippet: RT-PCR analysis of IDO mRNA expression in dTHP-1 cells and MdM stimulated for 24 h with C. trachomatis -infected HeLa cell supernatants and in dTHP-1 cells cocultivated with infected HeLa cells for 24 and 48 h. The cDNAs were amplified with IDO (324 bp, top panel) and GAPDH (306 bp, bottom panel) primers for 35 and 22 PCR cycles, respectively. A total of 10 μl of PCR products was loaded on a 2% agarose gel as follows. dTHP-1 cells and MdM were incubated with supernatants from HeLa cells left uninfected (lanes 1 and 6, respectively), infected with serovar E (lanes 2 and 7, respectively) or serovar L2 (lanes 3 and 8, respectively), treated with RPMI alone (lanes 4 and 9, respectively) or E. coli LPS alone (lanes 5 and 10, respectively), or dTHP-1 cells were incubated in coculture for 24 and 48 h with uninfected HeLa cells (lanes 11 and 14, respectively), with serovar E-infected HeLa cells (lanes 12 and 15, respectively), or with serovar L2-infected HeLa cells (lanes 13 and 16, respectively). A negative control for amplification (lane 17) wherein DNA was omitted and a positive control (lane 18) consisting of cDNA from HeLa cells exposed to rhIFN-γ (10 ng/ml for 12 h) were also included in the analysis.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Incubation, Negative Control, Positive Control

    36) Product Images from "Cloning and Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum islandicum †"

    Article Title: Cloning and Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum islandicum †

    Journal: Journal of Bacteriology

    doi:

    Application of DNA polymerase from P. islandicum in PCR. Reactions were carried out in a buffer containing 15 mM Tris-HCl (pH 8.6), 12.5 mM KCl, 2.5 mM (NH 4 ) 2 SO 4 , 1.25 mM MgCl 2 , and 20 μg of BSA per ml. One unit of DNA polymerase from P. islandicum was used to amplify 500 bp (lane 1), 1,000 bp (lane 2), and 1,500 bp (lane 3) of a λ DNA template. Control PCR was performed with 2.5 U of High Fidelity enzyme (lane 4). Twenty microliters of each PCR product was applied on an 1% agarose gel. The corresponding molecular sizes of the marker (lane 5) are indicated in kilobase pairs.
    Figure Legend Snippet: Application of DNA polymerase from P. islandicum in PCR. Reactions were carried out in a buffer containing 15 mM Tris-HCl (pH 8.6), 12.5 mM KCl, 2.5 mM (NH 4 ) 2 SO 4 , 1.25 mM MgCl 2 , and 20 μg of BSA per ml. One unit of DNA polymerase from P. islandicum was used to amplify 500 bp (lane 1), 1,000 bp (lane 2), and 1,500 bp (lane 3) of a λ DNA template. Control PCR was performed with 2.5 U of High Fidelity enzyme (lane 4). Twenty microliters of each PCR product was applied on an 1% agarose gel. The corresponding molecular sizes of the marker (lane 5) are indicated in kilobase pairs.

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

    37) Product Images from "Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA"

    Article Title: Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000965

    pUL29/28 specifically activates the MIE promoter irrespective of HCMV pUL38 expression. (A) pUL29/28 activates the MIEP. Upper left panel: U2OS cells that stably maintained the empty pLXSN vector (U2OS) or pLXSN expressing HCMV pUL38 (USOS-38) were transfected with 50 ng of pGL3-MIEP reporter plasmid and 10, 100, or 500 ng of pCGN empty vector or pCGN-pUL29/28 effector plasmid. Luciferase activity was assayed 48 h posttransfection using equal protein amounts within each lysate and normalized to luciferase activity from empty vector. Lower panels: The levels of pUL29/28HA and pUL38 expression were assayed by Western blot analysis using the same lysates and antibody to HA, pUL38 or tubulin. Right panel: UL29 and luciferase RNA expression was determined by qRT-PCR from U2OS as compared to U2OS-38 cells. (B) pUL29/28 exhibits promoter-specific effects. Luciferase assays were completed using 500 ng of pCGN or pCGN-pUL29/28 and MIEP, ISRE and NF-κB promoter reporter constructs. The relative luciferase activity was determined as described above. (C) NuRD is required for optimal expression of an MIEP reporter. Left panel: Disruption of the NuRD complex in U-2 OS cells. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4 or RBBP4 were delivered to U-2 OS cells using lentiviruses and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR. The data was normalized to GAPDH RNA levels and includes the percent reduction for each gene relative to control. Right panel: Luciferase assays were completed using either empty pCGN or pCGN-pUL29/28 and MIEP reporter. The relative luciferase activity was determined as described above and the data is derived from replicate experiments (**p
    Figure Legend Snippet: pUL29/28 specifically activates the MIE promoter irrespective of HCMV pUL38 expression. (A) pUL29/28 activates the MIEP. Upper left panel: U2OS cells that stably maintained the empty pLXSN vector (U2OS) or pLXSN expressing HCMV pUL38 (USOS-38) were transfected with 50 ng of pGL3-MIEP reporter plasmid and 10, 100, or 500 ng of pCGN empty vector or pCGN-pUL29/28 effector plasmid. Luciferase activity was assayed 48 h posttransfection using equal protein amounts within each lysate and normalized to luciferase activity from empty vector. Lower panels: The levels of pUL29/28HA and pUL38 expression were assayed by Western blot analysis using the same lysates and antibody to HA, pUL38 or tubulin. Right panel: UL29 and luciferase RNA expression was determined by qRT-PCR from U2OS as compared to U2OS-38 cells. (B) pUL29/28 exhibits promoter-specific effects. Luciferase assays were completed using 500 ng of pCGN or pCGN-pUL29/28 and MIEP, ISRE and NF-κB promoter reporter constructs. The relative luciferase activity was determined as described above. (C) NuRD is required for optimal expression of an MIEP reporter. Left panel: Disruption of the NuRD complex in U-2 OS cells. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4 or RBBP4 were delivered to U-2 OS cells using lentiviruses and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR. The data was normalized to GAPDH RNA levels and includes the percent reduction for each gene relative to control. Right panel: Luciferase assays were completed using either empty pCGN or pCGN-pUL29/28 and MIEP reporter. The relative luciferase activity was determined as described above and the data is derived from replicate experiments (**p

    Techniques Used: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Western Blot, RNA Expression, Quantitative RT-PCR, Construct, shRNA, Isolation, Derivative Assay

    38) Product Images from "The ORF61 Protein Encoded by Simian Varicella Virus and Varicella-Zoster Virus Inhibits NF-κB Signaling by Interfering with IκBα Degradation"

    Article Title: The ORF61 Protein Encoded by Simian Varicella Virus and Varicella-Zoster Virus Inhibits NF-κB Signaling by Interfering with IκBα Degradation

    Journal: Journal of Virology

    doi: 10.1128/JVI.01149-15

    SVV ORF61 is not required for the inhibition of NF-κB signaling and Snail accumulation. TRFs were mock infected or infected with SVV wt or an ORF61 deletion mutant (Δ61) at a 5:1 ratio for 48 h. (A) PCR was performed on DNA extracted from
    Figure Legend Snippet: SVV ORF61 is not required for the inhibition of NF-κB signaling and Snail accumulation. TRFs were mock infected or infected with SVV wt or an ORF61 deletion mutant (Δ61) at a 5:1 ratio for 48 h. (A) PCR was performed on DNA extracted from

    Techniques Used: Inhibition, Infection, Mutagenesis, Polymerase Chain Reaction

    39) Product Images from "Cooperation between Viral Interferon Regulatory Factor 4 and RTA To Activate a Subset of Kaposi's Sarcoma-Associated Herpesvirus Lytic Promoters"

    Article Title: Cooperation between Viral Interferon Regulatory Factor 4 and RTA To Activate a Subset of Kaposi's Sarcoma-Associated Herpesvirus Lytic Promoters

    Journal: Journal of Virology

    doi: 10.1128/JVI.00694-11

    vIRF4 contributes to KSHV reactivation. (A) KSHV latently infected iSLK.219 cells were either mock treated or transfected with empty vector or vector expressing full-length vIRF4. The next day, the transfected cultures were induced with 100 ng/ml doxycycline (DOX) to drive expression of a DOX-regulated RTA cDNA stably integrated into the cell genome and maintained for 72 h. Culture medium was collected, filtered to remove debris, including cells, and used to infect 293-PAN-Luc reporter cells. After 48 h, lysates were prepared and assayed for luciferase activity. Values represent the means and standard errors of the means of three independent transfections. (B) iSLK.219 cells were transfected with dsiRNAs against EGFP or K10/vIRF4 mRNA sequences. Cultures were induced to reactivate by using doxycycline, and RNA was harvested 72 h later and analyzed by quantitative RT-PCR using primers to detect K9/vIRF1 (open bars) and K10/vIRF4 (filled bars). (C) Results of an experiment similar to that shown in panel B, except that the culture medium was collected from induced and uninduced iSLK.219 cells and assayed for infectious KSHV virions by using 293-PAN-Luc cells. Values are expressed relative to the averages of the uninduced samples.
    Figure Legend Snippet: vIRF4 contributes to KSHV reactivation. (A) KSHV latently infected iSLK.219 cells were either mock treated or transfected with empty vector or vector expressing full-length vIRF4. The next day, the transfected cultures were induced with 100 ng/ml doxycycline (DOX) to drive expression of a DOX-regulated RTA cDNA stably integrated into the cell genome and maintained for 72 h. Culture medium was collected, filtered to remove debris, including cells, and used to infect 293-PAN-Luc reporter cells. After 48 h, lysates were prepared and assayed for luciferase activity. Values represent the means and standard errors of the means of three independent transfections. (B) iSLK.219 cells were transfected with dsiRNAs against EGFP or K10/vIRF4 mRNA sequences. Cultures were induced to reactivate by using doxycycline, and RNA was harvested 72 h later and analyzed by quantitative RT-PCR using primers to detect K9/vIRF1 (open bars) and K10/vIRF4 (filled bars). (C) Results of an experiment similar to that shown in panel B, except that the culture medium was collected from induced and uninduced iSLK.219 cells and assayed for infectious KSHV virions by using 293-PAN-Luc cells. Values are expressed relative to the averages of the uninduced samples.

    Techniques Used: Infection, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Luciferase, Activity Assay, Quantitative RT-PCR

    40) Product Images from "MDP-NOD2 stimulation induces HNP-1 secretion which contributes to NOD2 anti-bacterial function"

    Article Title: MDP-NOD2 stimulation induces HNP-1 secretion which contributes to NOD2 anti-bacterial function

    Journal: Inflammatory bowel diseases

    doi: 10.1002/ibd.21144

    NOD2 activation by MDP-LD increased hnp-1 mRNA level. hnp-1 mRNA level was measured using quantitative RT-PCR and normalized for gapdh mRNA after 24 h of stimulation with 1μg of MDP-LD in NOD2 expressing HCT116 and SW480 cells (A,B) and non-NOD2 expressing cell lines HEK293 and Caco-2 (C,D). *p
    Figure Legend Snippet: NOD2 activation by MDP-LD increased hnp-1 mRNA level. hnp-1 mRNA level was measured using quantitative RT-PCR and normalized for gapdh mRNA after 24 h of stimulation with 1μg of MDP-LD in NOD2 expressing HCT116 and SW480 cells (A,B) and non-NOD2 expressing cell lines HEK293 and Caco-2 (C,D). *p

    Techniques Used: Activation Assay, Quantitative RT-PCR, Expressing

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    Article Snippet: .. Comparison of Plasmodium malariae MSP119 sequences from other geographic locations Ten nanograms of DNA from P. malariae strains Greece I, Guyana, and Uganda I were PCR amplified using the forward and reverse long deoxyoligonucleotides described above and the Expand High Fidelity PCR system (Roche Applied Science, Indianapolis, IN, USA). .. Cycle conditions were as follows: 94 °C for 5 min, 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min, and a final extension step of 68 °C for 5 min. Products were purified (StrataPrep PCR purification kit, Stratagene) and sequenced as described above.

    Synthesized:

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Isolation:

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Quantitative RT-PCR:

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Real-time Polymerase Chain Reaction:

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

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    Article Snippet: .. Comparison of Plasmodium malariae MSP119 sequences from other geographic locations Ten nanograms of DNA from P. malariae strains Greece I, Guyana, and Uganda I were PCR amplified using the forward and reverse long deoxyoligonucleotides described above and the Expand High Fidelity PCR system (Roche Applied Science, Indianapolis, IN, USA). .. Cycle conditions were as follows: 94 °C for 5 min, 35 cycles of 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 1 min, and a final extension step of 68 °C for 5 min. Products were purified (StrataPrep PCR purification kit, Stratagene) and sequenced as described above.

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    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
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    Roche expand high fidelity polymerase chain reaction pcr system
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    91
    Roche hi fi polymerase chain reaction pcr taq polymerase
    Hi Fi Polymerase Chain Reaction Pcr Taq Polymerase, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hi fi polymerase chain reaction pcr taq polymerase/product/Roche
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hi fi polymerase chain reaction pcr taq polymerase - by Bioz Stars, 2020-09
    91/100 stars
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