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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 "Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation"

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

    Journal: Genome Medicine

    doi: 10.1186/s13073-018-0589-3

    T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control
    Figure Legend Snippet: T helper cells upregulate the IRE1a-XBP1 pathway during activation. a Schematic representation of the hypothesis. In this study, we are asking what role does the IRE1a-XBP1 pathway play during T helper cell activation. T helper cell activation is a dramatic transformation from a quiescent cell state to a rapidly proliferative and highly protein productive/secretive cellular state. b Overview of the experiment. Splenic naïve T cells were purified by negative selection and activated in anti-CD3e/C28 antibody-coated plates under Th2 differentiation conditions (i.e., in the presence of anti-IFNγ neutralizing antibody, IL2, and IL4) for 72 h, rested for 42 h, and restimulated on anti-CD3e/CD28 antibody-coated plate. Restimulated Th2 cells were used in RNA sequencing, ChIPmentation (ChIP sequencing), Western blot, qPCR, and flow cytometry. To perturb IRE1a-XBP1 pathway, we used 15-μM 4μ8c that specifically blocks the pathway by inhibiting IRE1a endonuclease activity. The drug was added to the culture media at the beginning of the culture and during passage from the activation plate to the resting plate. c Naïve T helper cells and in vitro differentiated Th2 lymphocytes were analyzed for IRE1a mRNA expression by qRT-PCR (left panel), protein expression by Western blot (middle panel), and phosphorylated IRE1a (P-IRE1a) by Western blot (right panel). The density of Western blot bands from five independent experiments of IRE1a and three independent experiments of phospho-IRE1a were measured and displayed on top of each Western blot panel. d Naïve T cells were cultured under Th2 differentiation conditions in the presence or absence of IRE1a inhibitor (4μ8c). In vitro reactivated Th2 lymphocytes were analyzed by RT-PCR using a pair of primers that discriminate the cDNA derived from spliced and unspliced form of XBP1 mRNA. Tunicamycin-treated Th2 cells were used as a positive control. e Naïve T helper cells (N) and in vitro differentiated and restimulated Th2 cells (differentiated in the presence or absence of 4μ8c) were stained with fluorescent dye-conjugated anti-XBP1s-specific antibody and analyzed by flow cytometry. Gating: singlets > live cells > XBP1s. One representative FACS profile is displayed (left panel), and the graph containing all results ( n = 5) is shown in the “right panel”. Tunicamycin-treated Th2 cells were used as a positive control

    Techniques Used: Activation Assay, Transformation Assay, Purification, Selection, RNA Sequencing Assay, Chromatin Immunoprecipitation, Sequencing, Western Blot, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Activity Assay, In Vitro, Expressing, Quantitative RT-PCR, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Positive Control, Staining, FACS

    4) Product Images from "Comparative mitochondrial genomics in zygomycetes: bacteria-like RNase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperms"

    Article Title: Comparative mitochondrial genomics in zygomycetes: bacteria-like RNase P RNAs, mobile elements and a close source of the group I intron invasion in angiosperms

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki199

    Secondary structure models for mtP-RNAs from R.oryzae , R.stolonifer 194667, R.oligosporus , R.spectabilis , M.mucedo , S.culisetae and M.verticillata . Positions in red are invariant in the minimum bacterial consensus ( 32 ); uppercase letters in the mtP-RNAs indicate 100%, lowercase 90%, conservation of the minimum bacterial consensus sequence. The arrows pinpoint experimentally determined termini; arrow length is proportional to the percentage of molecules ending at a defined position. Double hairpin elements are named in green. The few nucleotides colored blue in the R.stolonifer mtP-RNA model are different in its close relative R.oryzae .
    Figure Legend Snippet: Secondary structure models for mtP-RNAs from R.oryzae , R.stolonifer 194667, R.oligosporus , R.spectabilis , M.mucedo , S.culisetae and M.verticillata . Positions in red are invariant in the minimum bacterial consensus ( 32 ); uppercase letters in the mtP-RNAs indicate 100%, lowercase 90%, conservation of the minimum bacterial consensus sequence. The arrows pinpoint experimentally determined termini; arrow length is proportional to the percentage of molecules ending at a defined position. Double hairpin elements are named in green. The few nucleotides colored blue in the R.stolonifer mtP-RNA model are different in its close relative R.oryzae .

    Techniques Used: Sequencing

    5) Product Images from "Maintaining mRNA Integrity during Decalcification of Mineralized Tissues"

    Article Title: Maintaining mRNA Integrity during Decalcification of Mineralized Tissues

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0058154

    Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.
    Figure Legend Snippet: Quantitative PCR. Total RNA extracted from tibiae decalicified with 0.5 M EDTA, RNA later /EDTA at pH 9.2 or RNA later /EDTA at pH5.2 was analyzed for Col2a1 (A) or Rpl10 (B) mRNA transcripts by q PCR using Sybr Green. The machine output (Roche LightCycler 480 II) of fluorescence at each PCR cycle number is shown.

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, SYBR Green Assay, Fluorescence

    Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.
    Figure Legend Snippet: Fluorescent immunohistochemistry of articular cartilage. Collagen II (A, B) and collagen VI (C, D) protein localization was unaffected by RNA later /EDTA, pH 5.2 decalcification (B, D) compared to conventional EDTA decalcification (A, C). Using both methods collagen II can be seen in both the pericellular and extracellular matrix, while collagen VI is predominantly localized to the pericellular matrix. DAPI was used as a nuclear stain. Scale bars = 50 µm.

    Techniques Used: Immunohistochemistry, Staining

    In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).
    Figure Legend Snippet: In situ hybridization for Prg4 and Col2a1 . Cryosections from tibia decalcified with EDTA (A,B) or RNA later /EDTA at pH5.2 (C–F) were hybridized with DIG-labeled Prg4 antisense (A,C) or sense (B,D) RNA probes, and against 35 S-labeled Col2a1 antisense (E) or sense (F) RNA probes for Col2a1 . Arrows show representative regions of target gene mRNA expression. Scale bars = 100 µm (A–D), 10 µm (E, F).

    Techniques Used: In Situ Hybridization, Labeling, Expressing

    Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.
    Figure Legend Snippet: Cartilage morphology after EDTA or RNA later /EDTA decalcification. Tibial epiphyses were decalcified for 72 hrs at 4°C with 0.5 M EDTA (A) or RNA later /10% EDTA at pH 5.2 and cryosections were stained with toluidine blue/fast green. The medial tibial plateau is shown. Cartilage morphology and aggrecan staining is preserved in the RNA later /10% EDTA, pH 5.2 decalcified samples. Scale bar = 100 µm.

    Techniques Used: Staining

    Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).
    Figure Legend Snippet: Analysis of RNA integrity. Microcapillary electrophoresis of total RNA isolated from whole tibial epiphysis (A, C, E) or cryosections of tibiae (B, D, F) decalcified with 0.5 M EDTA (A, B), RNA later /EDTA at pH 9.2 (C, D) or RNA later /EDTA at pH5.2 (E, F).

    Techniques Used: Electrophoresis, Isolation

    6) Product Images from "Cellular transformation by combined lineage conversion and oncogene expression"

    Article Title: Cellular transformation by combined lineage conversion and oncogene expression

    Journal: bioRxiv

    doi: 10.1101/525600

    Generating proliferative induced hepatocytes using defined transcription factors and oncogenic drivers ( A ) Schematic outline of the cell transformation assay for making lineage-specific cancer by lentiviral expression of three lineage-specific TFs to convert HFs to induced hepatocytes (iHep) and defined oncogenic drivers to transform iHeps to proliferating and tumorigenic cells. ( B ) Comparison of TF combinations ( Du et al , 2014 , Huang et al , 2014 , Morris et al , 2014 ) for converting human fibroblasts to iHeps by detecting transcript levels for liver marker genes ( ALBUMIN , TRANSFERRIN and SERPINA1/α-1-antitrypsin ) by qRT-PCR at different time points after iHep conversion, normalized to GAPDH levels (mean ± standard error).
    Figure Legend Snippet: Generating proliferative induced hepatocytes using defined transcription factors and oncogenic drivers ( A ) Schematic outline of the cell transformation assay for making lineage-specific cancer by lentiviral expression of three lineage-specific TFs to convert HFs to induced hepatocytes (iHep) and defined oncogenic drivers to transform iHeps to proliferating and tumorigenic cells. ( B ) Comparison of TF combinations ( Du et al , 2014 , Huang et al , 2014 , Morris et al , 2014 ) for converting human fibroblasts to iHeps by detecting transcript levels for liver marker genes ( ALBUMIN , TRANSFERRIN and SERPINA1/α-1-antitrypsin ) by qRT-PCR at different time points after iHep conversion, normalized to GAPDH levels (mean ± standard error).

    Techniques Used: Cell Transformation Assay, Expressing, Marker, Quantitative RT-PCR

    7) Product Images from "Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo"

    Article Title: Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148777

    Preparation, appearance and gene expressions of aggregates of human BM MSCs after hanging drop culture. (A) Schema for the study. 2.5 × 10 5 human BM MSCs were cultured for 3 days in hanging drops. (B) Drops hanging on the cover of a 15 cm dish. (C) Macroscopic images of aggregates consisting of 2.5×10 5 MSCs at 10 minutes-72 hours. (D) Histological sections stained with Toluidine Blue at 72 hours. (E) TEM images of aggregates at 72 hours. (F) Histological sections immunostained with lubricin at 72 hours. (G) Real time PCR analysis for MSCs 72 hours after monolayer and pellet culture. RNA was prepared from 500,000 MSCs or 2 aggregates from 5 donors respectively. Gene expression levels in MSCs after monolayer culture were normalized as 1 (n = 5, *p
    Figure Legend Snippet: Preparation, appearance and gene expressions of aggregates of human BM MSCs after hanging drop culture. (A) Schema for the study. 2.5 × 10 5 human BM MSCs were cultured for 3 days in hanging drops. (B) Drops hanging on the cover of a 15 cm dish. (C) Macroscopic images of aggregates consisting of 2.5×10 5 MSCs at 10 minutes-72 hours. (D) Histological sections stained with Toluidine Blue at 72 hours. (E) TEM images of aggregates at 72 hours. (F) Histological sections immunostained with lubricin at 72 hours. (G) Real time PCR analysis for MSCs 72 hours after monolayer and pellet culture. RNA was prepared from 500,000 MSCs or 2 aggregates from 5 donors respectively. Gene expression levels in MSCs after monolayer culture were normalized as 1 (n = 5, *p

    Techniques Used: Cell Culture, Staining, Transmission Electron Microscopy, Real-time Polymerase Chain Reaction, Expressing

    8) Product Images from "Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation"

    Article Title: Acanthamoeba containing endosymbiotic chlamydia isolated from hospital environments and its potential role in inflammatory exacerbation

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0906-1

    Growth of Protochlamydia W-9 (W9) and Parachlamydia Bn 9 (Pac) in Acanthamoeba C3. a Representative images of DAPI staining showing Protochlamydia W9-infected C3 amoebae (Day 5 post-infection) and Parachlamydia Bn 9 -infected C3 amoebae (Day 3 post-infection). Arrows show bacterial clusters in each amoeba. b Changes in the number of chlamydial 16S rRNA copies per well. The data represent the average number of copies ± SD. The bacterial 16S rRNA copy numbers were normalized with the amoebal 18S rRNA copy numbers. *indicates p
    Figure Legend Snippet: Growth of Protochlamydia W-9 (W9) and Parachlamydia Bn 9 (Pac) in Acanthamoeba C3. a Representative images of DAPI staining showing Protochlamydia W9-infected C3 amoebae (Day 5 post-infection) and Parachlamydia Bn 9 -infected C3 amoebae (Day 3 post-infection). Arrows show bacterial clusters in each amoeba. b Changes in the number of chlamydial 16S rRNA copies per well. The data represent the average number of copies ± SD. The bacterial 16S rRNA copy numbers were normalized with the amoebal 18S rRNA copy numbers. *indicates p

    Techniques Used: Staining, Infection

    9) Product Images from "Computational Detection and Functional Analysis of Human Tissue-Specific A-to-I RNA Editing"

    Article Title: Computational Detection and Functional Analysis of Human Tissue-Specific A-to-I RNA Editing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018129

    Experimental validation of the predicted brain-specific and ovary-specific RNA editing sites. (A) The up/downstream region of the brain-specific RNA editing site was amplified successfully from cDNAs and gDNA of two adjacent non-cancerous brain tissues, as well as the HepG2, K562, MDA-MB-231, 293T, Raji, SW480 and SF126 cell lines. (B) Sequencing results of paired genomic DNA (control) and cDNA from the same human brain specimens and seven human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in both adjacent non-cancerous brain tissues and the human glioma cell line SF126. (C) The up/downstream region of the ovary-specific RNA editing site was amplified successfully from cDNAs and gDNA of the OVCAR3, SKOV3, SF126, HepG2, Raji, 293T, SW480, U2OS, K562 and MDA-MB-231 cell lines. (D) Sequencing results of paired genomic DNA (control) and cDNA from the same ten human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in the two human ovarian cancer cell lines SKOV3 and OVCAR3.
    Figure Legend Snippet: Experimental validation of the predicted brain-specific and ovary-specific RNA editing sites. (A) The up/downstream region of the brain-specific RNA editing site was amplified successfully from cDNAs and gDNA of two adjacent non-cancerous brain tissues, as well as the HepG2, K562, MDA-MB-231, 293T, Raji, SW480 and SF126 cell lines. (B) Sequencing results of paired genomic DNA (control) and cDNA from the same human brain specimens and seven human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in both adjacent non-cancerous brain tissues and the human glioma cell line SF126. (C) The up/downstream region of the ovary-specific RNA editing site was amplified successfully from cDNAs and gDNA of the OVCAR3, SKOV3, SF126, HepG2, Raji, 293T, SW480, U2OS, K562 and MDA-MB-231 cell lines. (D) Sequencing results of paired genomic DNA (control) and cDNA from the same ten human cell lines. A mixed peak of A and G in the cDNA sample but not in the genomic counterpart indicates the presence of RNA editing in the two human ovarian cancer cell lines SKOV3 and OVCAR3.

    Techniques Used: Amplification, Multiple Displacement Amplification, Sequencing

    10) Product Images from "Evidence for a Multiprotein Gamma-2 Herpesvirus Entry Complex ▿"

    Article Title: Evidence for a Multiprotein Gamma-2 Herpesvirus Entry Complex ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01141-07

    gp150 and gL are both dispensable for latency establishment in vivo. (A) BALB/c mice were infected intranasally with eGFP − MHV-68 mutants as shown. Thirteen days later, the viral load in spleens was measured by infectious center assay. Each point
    Figure Legend Snippet: gp150 and gL are both dispensable for latency establishment in vivo. (A) BALB/c mice were infected intranasally with eGFP − MHV-68 mutants as shown. Thirteen days later, the viral load in spleens was measured by infectious center assay. Each point

    Techniques Used: In Vivo, Mouse Assay, Infection

    gp150 inhibits and gL does not affect MHV-68 binding to splenic B cells. BHK-21 fibroblasts, NMuMG epithelial cells, and CD19 + splenic B cells were exposed to gM-eGFP + versions of the gL and gp150 mutants for different times and at different
    Figure Legend Snippet: gp150 inhibits and gL does not affect MHV-68 binding to splenic B cells. BHK-21 fibroblasts, NMuMG epithelial cells, and CD19 + splenic B cells were exposed to gM-eGFP + versions of the gL and gp150 mutants for different times and at different

    Techniques Used: Binding Assay

    The gB/gH association does not extend to all forms of gH but is independent of gL, gp150, and gp70. (A) gH was immunoprecipitated (IP) from lysates of wild-type (WT) or gL-deficient (gL − ) virions (the gL − DEL-STOP mutant) with MAbs specific
    Figure Legend Snippet: The gB/gH association does not extend to all forms of gH but is independent of gL, gp150, and gp70. (A) gH was immunoprecipitated (IP) from lysates of wild-type (WT) or gL-deficient (gL − ) virions (the gL − DEL-STOP mutant) with MAbs specific

    Techniques Used: Immunoprecipitation, Mutagenesis

    Viral gp150 expression depends in part on gL. (A) gL + virions—the wild type (WT), the gL − DEL revertant (rev), and the gL − STOP revertant—were compared with three independent gL mutants, i.e., gL − DEL, gL
    Figure Legend Snippet: Viral gp150 expression depends in part on gL. (A) gL + virions—the wild type (WT), the gL − DEL revertant (rev), and the gL − STOP revertant—were compared with three independent gL mutants, i.e., gL − DEL, gL

    Techniques Used: Expressing

    The gp150 disruption and gL disruption phenotypes are distinct. (A) The gL − DEL-STOP (gL − ) and gp150 − STOP (gp150 − ) mutations were transferred individually and together onto a gM-eGFP background, in which virions are fluorescent
    Figure Legend Snippet: The gp150 disruption and gL disruption phenotypes are distinct. (A) The gL − DEL-STOP (gL − ) and gp150 − STOP (gp150 − ) mutations were transferred individually and together onto a gM-eGFP background, in which virions are fluorescent

    Techniques Used:

    11) Product Images from "Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis"

    Article Title: Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis

    Journal: World Journal of Biological Chemistry

    doi: 10.4331/wjbc.v6.i3.249

    Growth of the wild-type and Prx-deficient ΔAhpC_H1, ΔAhpC_H2, ΔB_BCP, and ΔB_TPx B. subtilis strains. A: Bacterial growth was monitored by the optical density at 600 nm (OD600) for up to 25 h after addition of the same
    Figure Legend Snippet: Growth of the wild-type and Prx-deficient ΔAhpC_H1, ΔAhpC_H2, ΔB_BCP, and ΔB_TPx B. subtilis strains. A: Bacterial growth was monitored by the optical density at 600 nm (OD600) for up to 25 h after addition of the same

    Techniques Used:

    12) 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

    13) Product Images from "CK1α overexpression correlates with poor survival in colorectal cancer"

    Article Title: CK1α overexpression correlates with poor survival in colorectal cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4019-0

    Tumor differentiation-dependent investigation of CK1α RNA expression. a Box plot representing group comparison of relative CK1α RNA expression in low-grade and high-grade CRC patients. b Kaplan-Meier plot displaying the overall survival of Grade 3 and Grade 4 tumors of CRC patients, divided according to relative CK1α RNA expression. *** p
    Figure Legend Snippet: Tumor differentiation-dependent investigation of CK1α RNA expression. a Box plot representing group comparison of relative CK1α RNA expression in low-grade and high-grade CRC patients. b Kaplan-Meier plot displaying the overall survival of Grade 3 and Grade 4 tumors of CRC patients, divided according to relative CK1α RNA expression. *** p

    Techniques Used: RNA Expression

    Impact of CK1α RNA expression on prognosis of different subgroups of CRC patients. a Kaplan-Meier plot displaying the overall survival of UICC II and UICC III tumors of CRC patients, divided according to relative CK1α RNA expression. b Kaplan-Meier plot displaying the overall survival of RCC patients, divided according to relative CK1α RNA expression. CK1α RNA expression in colorectal tumor tissue of all grades was relatively quantified by qPCR using specific primers. HPRT gene served as reference gene. Graphs were created using IBM SPSS Statistics 20
    Figure Legend Snippet: Impact of CK1α RNA expression on prognosis of different subgroups of CRC patients. a Kaplan-Meier plot displaying the overall survival of UICC II and UICC III tumors of CRC patients, divided according to relative CK1α RNA expression. b Kaplan-Meier plot displaying the overall survival of RCC patients, divided according to relative CK1α RNA expression. CK1α RNA expression in colorectal tumor tissue of all grades was relatively quantified by qPCR using specific primers. HPRT gene served as reference gene. Graphs were created using IBM SPSS Statistics 20

    Techniques Used: RNA Expression, Real-time Polymerase Chain Reaction

    Investigation of CK1α RNA expression as a prognostic marker in CRC. a Box plot representing group comparison of relative CK1α RNA expression in normal and tumor tissue of CRC patients. b Kaplan-Meier plot displaying the overall survival of whole cohort, divided according to relative CK1α RNA expression in tumor tissue. * p
    Figure Legend Snippet: Investigation of CK1α RNA expression as a prognostic marker in CRC. a Box plot representing group comparison of relative CK1α RNA expression in normal and tumor tissue of CRC patients. b Kaplan-Meier plot displaying the overall survival of whole cohort, divided according to relative CK1α RNA expression in tumor tissue. * p

    Techniques Used: RNA Expression, Marker

    14) Product Images from "Evaluation of Macaca radiata as a non-human primate model of Dengue virus infection"

    Article Title: Evaluation of Macaca radiata as a non-human primate model of Dengue virus infection

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-21582-9

    Phylogenetic analysis of DENV 09-48 strain. A phylogenetic tree was constructed using the maximum likelihood method with the DENV-4 polyprotein coding region. The DENV-4 09-48 strain is indicated by the black triangle. The country and year of isolation as well as the accession number are indicated.
    Figure Legend Snippet: Phylogenetic analysis of DENV 09-48 strain. A phylogenetic tree was constructed using the maximum likelihood method with the DENV-4 polyprotein coding region. The DENV-4 09-48 strain is indicated by the black triangle. The country and year of isolation as well as the accession number are indicated.

    Techniques Used: Construct, Isolation

    15) Product Images from "Interaction between Leukotoxin and Cu,Zn Superoxide Dismutase in Aggregatibacter actinomycetemcomitans ▿"

    Article Title: Interaction between Leukotoxin and Cu,Zn Superoxide Dismutase in Aggregatibacter actinomycetemcomitans ▿

    Journal:

    doi: 10.1128/IAI.00288-07

    Site-directed mutagenesis of sodC . (A) PCR product of sodC gene. Lanes: 1, sodC ::EZ-Tn5; 2, wild-type sodC . The sizes on the left are in bp. (B) Western blot analysis of Cu,Zn SOD in A. actinomycetemcomitans . Lanes: 1, strain DF2200; 2, sodC mutant strain
    Figure Legend Snippet: Site-directed mutagenesis of sodC . (A) PCR product of sodC gene. Lanes: 1, sodC ::EZ-Tn5; 2, wild-type sodC . The sizes on the left are in bp. (B) Western blot analysis of Cu,Zn SOD in A. actinomycetemcomitans . Lanes: 1, strain DF2200; 2, sodC mutant strain

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Western Blot

    16) Product Images from "Mutant IDH1 Confers an in Vivo Growth in a Melanoma Cell Line with BRAF Mutation"

    Article Title: Mutant IDH1 Confers an in Vivo Growth in a Melanoma Cell Line with BRAF Mutation

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2010.12.011

    Growth advantage by mutant IDH1 in BRAF -mutated melanoma cells. A : Immunoblot analysis of mock, wild-type IDH1 (WT1 and 2), and mutant IDH1 (MUT1 and 2). The FLAG-tagged wild or mutant (R132H) IDH1 genes were introduced in BRAF -mutated G361 cells. The
    Figure Legend Snippet: Growth advantage by mutant IDH1 in BRAF -mutated melanoma cells. A : Immunoblot analysis of mock, wild-type IDH1 (WT1 and 2), and mutant IDH1 (MUT1 and 2). The FLAG-tagged wild or mutant (R132H) IDH1 genes were introduced in BRAF -mutated G361 cells. The

    Techniques Used: Mutagenesis

    17) Product Images from "The Antifungal Vaccine Derived from the Recombinant N Terminus of Als3p Protects Mice against the Bacterium Staphylococcus aureus ▿"

    Article Title: The Antifungal Vaccine Derived from the Recombinant N Terminus of Als3p Protects Mice against the Bacterium Staphylococcus aureus ▿

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00700-08

    Candidal rAls3p-N vaccine primes murine splenocytes against the S. aureus cell wall. Splenocytes from mice vaccinated with CFA alone or CFA plus rAls3p-N were stimulated with medium alone, tetanus toxoid, rAls3p-N, S. aureus cell wall extract, or rClfA-N.
    Figure Legend Snippet: Candidal rAls3p-N vaccine primes murine splenocytes against the S. aureus cell wall. Splenocytes from mice vaccinated with CFA alone or CFA plus rAls3p-N were stimulated with medium alone, tetanus toxoid, rAls3p-N, S. aureus cell wall extract, or rClfA-N.

    Techniques Used: Mouse Assay

    18) Product Images from "miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells"

    Article Title: miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells

    Journal: Journal of Cell Communication and Signaling

    doi: 10.1007/s12079-017-0410-x

    miR-217-5p expression is increased upon induction of the apoptosis. For validation screening HCT 116, SKOV3, and T98G cells were seeded 24 h before transfection with miRNA mimics (50 nM and 0.4 μl ScreenFect®A) or non-targeting siRNA (NT) control. Apoptosis rates 72 h after transfection were analyzed by Nicoletti staining followed by flow cytometric analysis ( a ). For miR-217-3p and -5p expression analysis total RNA was isolated from untreated HCT 116, HT-29 and SW480 cells and applied to cDNA synthesis followed by qRT-PCR. The expression analysis of both miR-217 strands was done by normalization to the CT value of U6 snRNA ( b ). For determination of miR-217-5p expression after induction of apoptosis, cells were seeded in 24 h prior treatment with Etoposide (25 μM), TRAIL (150 ng/ml, except HCT 116 with 80 ng/ml) or DMSO for additional 48 h. The apoptosis rates 48 h after treatment were analyzed by Nicoletti staining and flow cytometric analysis ( c ). The miRNA expression of miR-217-5p was normalized to the CT value of U6 snRNA and the untreated control ( d ). Statistical analyses for part ( a , c and d ) were performed by two-way ANOVA followed by Bonferroni post-test. Statistical differences for part B were tested using unpaired t-test. The treatments were compared to NT ( a ) or untreated cells ( c and d ) [ n = 3 biological replicates; mean ± SD, * p
    Figure Legend Snippet: miR-217-5p expression is increased upon induction of the apoptosis. For validation screening HCT 116, SKOV3, and T98G cells were seeded 24 h before transfection with miRNA mimics (50 nM and 0.4 μl ScreenFect®A) or non-targeting siRNA (NT) control. Apoptosis rates 72 h after transfection were analyzed by Nicoletti staining followed by flow cytometric analysis ( a ). For miR-217-3p and -5p expression analysis total RNA was isolated from untreated HCT 116, HT-29 and SW480 cells and applied to cDNA synthesis followed by qRT-PCR. The expression analysis of both miR-217 strands was done by normalization to the CT value of U6 snRNA ( b ). For determination of miR-217-5p expression after induction of apoptosis, cells were seeded in 24 h prior treatment with Etoposide (25 μM), TRAIL (150 ng/ml, except HCT 116 with 80 ng/ml) or DMSO for additional 48 h. The apoptosis rates 48 h after treatment were analyzed by Nicoletti staining and flow cytometric analysis ( c ). The miRNA expression of miR-217-5p was normalized to the CT value of U6 snRNA and the untreated control ( d ). Statistical analyses for part ( a , c and d ) were performed by two-way ANOVA followed by Bonferroni post-test. Statistical differences for part B were tested using unpaired t-test. The treatments were compared to NT ( a ) or untreated cells ( c and d ) [ n = 3 biological replicates; mean ± SD, * p

    Techniques Used: Expressing, Transfection, Staining, Flow Cytometry, Isolation, Quantitative RT-PCR

    19) Product Images from "The CDK Inhibitor p21Cip1/WAF1 Is Induced by Fc?R Activation and Restricts the Replication of Human Immunodeficiency Virus Type 1 and Related Primate Lentiviruses in Human Macrophages ▿"

    Article Title: The CDK Inhibitor p21Cip1/WAF1 Is Induced by Fc?R Activation and Restricts the Replication of Human Immunodeficiency Virus Type 1 and Related Primate Lentiviruses in Human Macrophages ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01395-09

    PMA and the HDAC inhibitor MS-275 induce p21 expression and inhibit HIV-1 replication in macrophages. (A) Macrophages were treated with PMA (30 and 100 ng/ml) and infected with HIV-1 VSV-G . The luciferase activity was measured 72 h p.i. For the inset,
    Figure Legend Snippet: PMA and the HDAC inhibitor MS-275 induce p21 expression and inhibit HIV-1 replication in macrophages. (A) Macrophages were treated with PMA (30 and 100 ng/ml) and infected with HIV-1 VSV-G . The luciferase activity was measured 72 h p.i. For the inset,

    Techniques Used: Mass Spectrometry, Expressing, Infection, Luciferase, Activity Assay

    p21 restricts HIV-1 reverse transcription and integration in macrophages. Macrophages were transfected with a p21-specific siRNA or an irrelevant siRNA (si-Neg) in the presence (S) or absence (US) of IC. Cells were infected with HIV-1 VSV-G 24 h after
    Figure Legend Snippet: p21 restricts HIV-1 reverse transcription and integration in macrophages. Macrophages were transfected with a p21-specific siRNA or an irrelevant siRNA (si-Neg) in the presence (S) or absence (US) of IC. Cells were infected with HIV-1 VSV-G 24 h after

    Techniques Used: Transfection, Infection

    Degradation of incoming viruses does not account for the defective HIV-1 reverse transcription. (A) Macrophages plated in untreated (unstimulated, US) or IC-coated plates (stimulated, S) were infected with HIV-1 VSV-G . Late reverse transcription products
    Figure Legend Snippet: Degradation of incoming viruses does not account for the defective HIV-1 reverse transcription. (A) Macrophages plated in untreated (unstimulated, US) or IC-coated plates (stimulated, S) were infected with HIV-1 VSV-G . Late reverse transcription products

    Techniques Used: Infection

    FcγR aggregation induces p21 protein expression specifically and irrespective of p53 expression. (A) Uninfected and HIV-1 VSV-G -infected macrophages were either left untreated (US) or stimulated with IC or LPS (100 ng/ml) for 48 h before total
    Figure Legend Snippet: FcγR aggregation induces p21 protein expression specifically and irrespective of p53 expression. (A) Uninfected and HIV-1 VSV-G -infected macrophages were either left untreated (US) or stimulated with IC or LPS (100 ng/ml) for 48 h before total

    Techniques Used: Expressing, Infection

    p21 silencing enhances HIV-1 replication in macrophages. (A) Macrophages were seeded in the presence (S) or absence (US) of IC and immediately transfected with p21-specific siRNA duplexes n.9 and n.12 or SMARTpool for p21 (Dharmacon), or a scrambled siRNA
    Figure Legend Snippet: p21 silencing enhances HIV-1 replication in macrophages. (A) Macrophages were seeded in the presence (S) or absence (US) of IC and immediately transfected with p21-specific siRNA duplexes n.9 and n.12 or SMARTpool for p21 (Dharmacon), or a scrambled siRNA

    Techniques Used: Transfection

    p21 protein interaction with viral components of the HIV-1 PIC is not detected in yeast two-hybrid or in vitro. (A) Two-hybrid assay between p21 and viral components of the PIC. The yeast reporter strain L40, expressing the indicated pairs of hybrid proteins,
    Figure Legend Snippet: p21 protein interaction with viral components of the HIV-1 PIC is not detected in yeast two-hybrid or in vitro. (A) Two-hybrid assay between p21 and viral components of the PIC. The yeast reporter strain L40, expressing the indicated pairs of hybrid proteins,

    Techniques Used: In Vitro, Two Hybrid Assay, Expressing

    20) Product Images from "Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain ▿"

    Article Title: Tissue-Specific Sequence Alterations in the Human Immunodeficiency Virus Type 1 Envelope Favoring CCR5 Usage Contribute to Persistence of Dual-Tropic Virus in the Brain ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02648-08

    Coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A and B) or MDM (C and D) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase
    Figure Legend Snippet: Coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A and B) or MDM (C and D) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase

    Techniques Used: Infection, Luciferase

    Coreceptor preference for HIV-1 entry into JC53 cells. JC53 cells were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase reporter virus,
    Figure Legend Snippet: Coreceptor preference for HIV-1 entry into JC53 cells. JC53 cells were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped luciferase reporter virus,

    Techniques Used: Infection, Luciferase

    Env V3 determinants influencing coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A) or MDM (B) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped
    Figure Legend Snippet: Env V3 determinants influencing coreceptor usage for HIV-1 entry into PBMC and MDM. PBMC (A) or MDM (B) were treated with maraviroc (1 μM), AMD3100 (1 μM), or both inhibitors prior to infection with equivalent infectious units of Env-pseudotyped

    Techniques Used: Infection

    21) Product Images from "Host Alternation of Chikungunya Virus Increases Fitness while Restricting Population Diversity and Adaptability to Novel Selective Pressures ▿Host Alternation of Chikungunya Virus Increases Fitness while Restricting Population Diversity and Adaptability to Novel Selective Pressures ▿ †Host Alternation of Chikungunya Virus Increases Fitness while Restricting Population Diversity and Adaptability to Novel Selective Pressures ▿ † ‡"

    Article Title: Host Alternation of Chikungunya Virus Increases Fitness while Restricting Population Diversity and Adaptability to Novel Selective Pressures ▿Host Alternation of Chikungunya Virus Increases Fitness while Restricting Population Diversity and Adaptability to Novel Selective Pressures ▿ †Host Alternation of Chikungunya Virus Increases Fitness while Restricting Population Diversity and Adaptability to Novel Selective Pressures ▿ † ‡

    Journal: Journal of Virology

    doi: 10.1128/JVI.01918-10

    Increases in genetic diversity are greatest when no alternating passages are imposed. CHIKV RNAs from supernatants of passages were reverse transcribed, and amplicons flanking the E1 gene were cloned into TOPO vectors and sequenced. The mutation frequency (average number of mutations per E1 gene) was calculated by dividing the number of nucleotide polymorphisms in all clones by the number of nucleotides sequenced and then multiplying by the CHIKV E1 length (1,317 nt). When the same polymorphism was present in multiple RNAs in the population, it was counted each time that it occurred. The E1 gene was sequenced a mean of 71 times (∼90,000 nucleotides/population). Each frequency was adjusted by subtracting the background error rate, estimated from mutations in TOPO-cloned CHIKV plasmid DNA. Numbers above the lines denote P values (χ 2 test with Yates' correction) comparing frequencies.
    Figure Legend Snippet: Increases in genetic diversity are greatest when no alternating passages are imposed. CHIKV RNAs from supernatants of passages were reverse transcribed, and amplicons flanking the E1 gene were cloned into TOPO vectors and sequenced. The mutation frequency (average number of mutations per E1 gene) was calculated by dividing the number of nucleotide polymorphisms in all clones by the number of nucleotides sequenced and then multiplying by the CHIKV E1 length (1,317 nt). When the same polymorphism was present in multiple RNAs in the population, it was counted each time that it occurred. The E1 gene was sequenced a mean of 71 times (∼90,000 nucleotides/population). Each frequency was adjusted by subtracting the background error rate, estimated from mutations in TOPO-cloned CHIKV plasmid DNA. Numbers above the lines denote P values (χ 2 test with Yates' correction) comparing frequencies.

    Techniques Used: Clone Assay, Mutagenesis, Plasmid Preparation

    Alternately passaged CHIKV populations contain more viable genomes than serially passaged populations. (A) The mutation frequency for molecular clones, representing all RNAs in the supernatant, was compared to that for RNAs isolated from plaques, representing only replication-competent RNAs. The molecular clone/plaque clone ratio (indicated above the bars) is calculated by dividing the mutation frequency in molecular clones by that for plaque clones from the same population. A mean of 91,189 (range, 41,773 to 141,805) nucleotides were sequenced for molecular or plaque clone RNAs. (B) The genome/PFU ratio was determined by dividing the number of CHIKV genomes measured by quantitative RT-PCR (4 replicates) by the number of PFU in passages of known titer, determined by plaque assay (duplicate titrations). Differences in ratios were compared using the χ 2 test with Yates' correction; line above the bars shows P value between populations.
    Figure Legend Snippet: Alternately passaged CHIKV populations contain more viable genomes than serially passaged populations. (A) The mutation frequency for molecular clones, representing all RNAs in the supernatant, was compared to that for RNAs isolated from plaques, representing only replication-competent RNAs. The molecular clone/plaque clone ratio (indicated above the bars) is calculated by dividing the mutation frequency in molecular clones by that for plaque clones from the same population. A mean of 91,189 (range, 41,773 to 141,805) nucleotides were sequenced for molecular or plaque clone RNAs. (B) The genome/PFU ratio was determined by dividing the number of CHIKV genomes measured by quantitative RT-PCR (4 replicates) by the number of PFU in passages of known titer, determined by plaque assay (duplicate titrations). Differences in ratios were compared using the χ 2 test with Yates' correction; line above the bars shows P value between populations.

    Techniques Used: Mutagenesis, Clone Assay, Isolation, Quantitative RT-PCR, Plaque Assay

    22) 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

    Human family of Mex-3 proteins. ( A ) Structure and chromosomal localization of the four human Mex -3 genes. Exons are depicted as boxes, with coding sequences as grey boxes and 3’-UTR as white boxes. Genes lying downstream and upstream of hMex -3 A, hMex -3 B, hMex -3 C and hMex -3 D genes are displayed as large arrows. ( B ) Sequence alignment of the hMex-3 proteins and their Caenorhabditis elegans ortholog. C. elegans Mex-3 and hMex-3 amino acid sequences were aligned with ClustalW. Identical residues (red) are annotated by an asterisk, whereas similar residues (green) and lightly similar residues (blue) are denoted with two or one dot, respectively. Junctions between the two exons (vertical line) lie in the same position for the four hMex -3 mRNA. One NES is predicted in each hMex-3 protein, and one NLS is also predicted for hMex-3B and hMex-3C. Two strongly conserved KH domains of type I were predicted in hMex-3 proteins. One conserved RING domain of C3H4 type was also predicted in C-terminus of the four human proteins. The highly conserved amino acids of KH and RING domains are shown as green boxes. Arrows indicate the amino acids which were mutated in this study. ( C ) Phylogenetic tree of the Mex-3 gene family. The 180 most conserved amino acids around the KH domains of human Mex-3, mouse Mex-3, ceMex-3 (Genbank accession number AAK73873) and two Ascidian Mex-3 homologous sequences from Ciona savignyi and Halocynthia roretzi (Genbank accession number BAB03404 and BAC10968) were aligned by T-coffee. A tree was done with PhyML method on this alignment. Bootstrap values are indicated as percentages (500 replicates). Scale bar: 0.05.
    Figure Legend Snippet: Human family of Mex-3 proteins. ( A ) Structure and chromosomal localization of the four human Mex -3 genes. Exons are depicted as boxes, with coding sequences as grey boxes and 3’-UTR as white boxes. Genes lying downstream and upstream of hMex -3 A, hMex -3 B, hMex -3 C and hMex -3 D genes are displayed as large arrows. ( B ) Sequence alignment of the hMex-3 proteins and their Caenorhabditis elegans ortholog. C. elegans Mex-3 and hMex-3 amino acid sequences were aligned with ClustalW. Identical residues (red) are annotated by an asterisk, whereas similar residues (green) and lightly similar residues (blue) are denoted with two or one dot, respectively. Junctions between the two exons (vertical line) lie in the same position for the four hMex -3 mRNA. One NES is predicted in each hMex-3 protein, and one NLS is also predicted for hMex-3B and hMex-3C. Two strongly conserved KH domains of type I were predicted in hMex-3 proteins. One conserved RING domain of C3H4 type was also predicted in C-terminus of the four human proteins. The highly conserved amino acids of KH and RING domains are shown as green boxes. Arrows indicate the amino acids which were mutated in this study. ( C ) Phylogenetic tree of the Mex-3 gene family. The 180 most conserved amino acids around the KH domains of human Mex-3, mouse Mex-3, ceMex-3 (Genbank accession number AAK73873) and two Ascidian Mex-3 homologous sequences from Ciona savignyi and Halocynthia roretzi (Genbank accession number BAB03404 and BAC10968) were aligned by T-coffee. A tree was done with PhyML method on this alignment. Bootstrap values are indicated as percentages (500 replicates). Scale bar: 0.05.

    Techniques Used: Sequencing

    Subcellular localization of hMex-3 proteins. ( A ) MCF7 cells expressing hMex-3A, -3B and -3C were stained with anti-myc antibody ( top ) or with specific anti-hM3Aβ or anti-hM3Bβ antibodies ( bottom ) and revealed by FITC-conjugated secondary antibodies. ( B ) MCF7 cells expressing hMex-3A, -3B, -3C proteins or hMex-3C mutated within the nuclear export signal (NES) sequence were treated with or without 20 ng/ml of Leptomycin B (LMB) and were stained as above. pEGFP-Rev-NES construct was used as a positive control. Scale bar: 20 μm.
    Figure Legend Snippet: Subcellular localization of hMex-3 proteins. ( A ) MCF7 cells expressing hMex-3A, -3B and -3C were stained with anti-myc antibody ( top ) or with specific anti-hM3Aβ or anti-hM3Bβ antibodies ( bottom ) and revealed by FITC-conjugated secondary antibodies. ( B ) MCF7 cells expressing hMex-3A, -3B, -3C proteins or hMex-3C mutated within the nuclear export signal (NES) sequence were treated with or without 20 ng/ml of Leptomycin B (LMB) and were stained as above. pEGFP-Rev-NES construct was used as a positive control. Scale bar: 20 μm.

    Techniques Used: Expressing, Staining, Sequencing, Construct, Positive Control

    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

    23) Product Images from "GapA and CrmA Coexpression Is Essential for Mycoplasma gallisepticum Cytadherence and Virulence "

    Article Title: GapA and CrmA Coexpression Is Essential for Mycoplasma gallisepticum Cytadherence and Virulence

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.12.6839-6845.2002

    Putative binding and interactive domains of M. gallisepticum GapA (A) and CrmA (B). [ ], transmembrane region (TM); [ ], putative signal peptidase cleavage site; [ ]).
    Figure Legend Snippet: Putative binding and interactive domains of M. gallisepticum GapA (A) and CrmA (B). [ ], transmembrane region (TM); [ ], putative signal peptidase cleavage site; [ ]).

    Techniques Used: Binding Assay

    Characterization of the M. gallisepticum R high transformants SDCA and GCA1 by immunoblotting and DNA hybridization analysis. Lane 1, R high ; lane 2, R low ; lane 3, SDCA c4 ; lane 4, GCA1 c5 . (A) Immunoblots developed with mixed anti-GapA and anti-CrmA sera. (B) Southern blot of Hin dIII-digested total genomic DNAs probed with a 32 P-labeled portion of crmA . Lanes in this digitized image have been reordered by using Adobe PhotoShop.
    Figure Legend Snippet: Characterization of the M. gallisepticum R high transformants SDCA and GCA1 by immunoblotting and DNA hybridization analysis. Lane 1, R high ; lane 2, R low ; lane 3, SDCA c4 ; lane 4, GCA1 c5 . (A) Immunoblots developed with mixed anti-GapA and anti-CrmA sera. (B) Southern blot of Hin dIII-digested total genomic DNAs probed with a 32 P-labeled portion of crmA . Lanes in this digitized image have been reordered by using Adobe PhotoShop.

    Techniques Used: DNA Hybridization, Western Blot, Southern Blot, Labeling

    24) 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

    25) Product Images from "High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene"

    Article Title: High-level Genomic Integration, Epigenetic Changes, and Expression of Sleeping Beauty Transgene

    Journal: Biochemistry

    doi: 10.1021/bi9016846

    Genomic copy number and chromosomal location of SB -IHK-DsRed insertions in individual K562 clones. ( A ) Genomic DNA isolated from single-cell clones was used as template for PCR using primers specific for DsRed and for the endogenous genomic HS3. ( B ) Determination of the SB -Tn insertion sites in cell clones. Using ligation-mediated nested PCR of the isolated genomic DNA, the insertion sites of the SB -IHK-DsRed were amplified and sequenced. NCBI BLAST analysis of the recovered flanking sequences was used to determine the genomic insertion sites in the K562 clones. The proportion of DsRed + cells in each individual clones was determined by FACS analysis. Number of insertion loci identified by ligation-mediated PCR corresponded with the number estimated by semi-quantitative PCR in panel ( A ). MS prefix represents cell clones isolated by FACS machine whereas other cell clones were isolated manually.
    Figure Legend Snippet: Genomic copy number and chromosomal location of SB -IHK-DsRed insertions in individual K562 clones. ( A ) Genomic DNA isolated from single-cell clones was used as template for PCR using primers specific for DsRed and for the endogenous genomic HS3. ( B ) Determination of the SB -Tn insertion sites in cell clones. Using ligation-mediated nested PCR of the isolated genomic DNA, the insertion sites of the SB -IHK-DsRed were amplified and sequenced. NCBI BLAST analysis of the recovered flanking sequences was used to determine the genomic insertion sites in the K562 clones. The proportion of DsRed + cells in each individual clones was determined by FACS analysis. Number of insertion loci identified by ligation-mediated PCR corresponded with the number estimated by semi-quantitative PCR in panel ( A ). MS prefix represents cell clones isolated by FACS machine whereas other cell clones were isolated manually.

    Techniques Used: Clone Assay, Isolation, Polymerase Chain Reaction, Ligation, Nested PCR, Amplification, FACS, Real-time Polymerase Chain Reaction, Mass Spectrometry

    DNA methylation at the inserted SB -Tns and endogenous host copy of ANKYRIN1 promoter. ( A ) COBRA analyses to establish overall CpG methylation levels at the investigated regions. Bisulfite PCR products digested with TaqI restriction endonuclease (T) were compared with uncut controls (N) for each samples of different SB clones. ( B ) Methylation at each CpG dinucleotide in the 620 bp region indicated under the schematic map was profiled by bisulfite-mediated genomic sequencing. Open circles represent unmethylated CpGs while filled circles are methylated CpGs. Each horizontal line of circles is derived from a single bisulfite PCR amplicon. Bisulfite-PCR amplicons derived from a cell clone were organized into each block with the clone number indicated at right. The end of the ANKYRIN1 promoter sequence and start of the DsRed CDS are indicated by the solid line through the blocks, with the above arrow indicating the direction of transcription. ( C ) CpG methylation profile at the endogenous host copy of ANKYRIN1 promoter. The region identical with ANKYRIN1 promoter sequence in the SB -IHK-DsRed is indicated in the schematic representation above the blocks of CpG methylation data derived from individual bisulfite-PCR amplicons. The cell clone from which genomic DNA was isolated is indicated at right, and methylated CpGs indicated by filled circles. The absence of CpG methylation at this region was also verified in the cell clone MS14 (data not shown).
    Figure Legend Snippet: DNA methylation at the inserted SB -Tns and endogenous host copy of ANKYRIN1 promoter. ( A ) COBRA analyses to establish overall CpG methylation levels at the investigated regions. Bisulfite PCR products digested with TaqI restriction endonuclease (T) were compared with uncut controls (N) for each samples of different SB clones. ( B ) Methylation at each CpG dinucleotide in the 620 bp region indicated under the schematic map was profiled by bisulfite-mediated genomic sequencing. Open circles represent unmethylated CpGs while filled circles are methylated CpGs. Each horizontal line of circles is derived from a single bisulfite PCR amplicon. Bisulfite-PCR amplicons derived from a cell clone were organized into each block with the clone number indicated at right. The end of the ANKYRIN1 promoter sequence and start of the DsRed CDS are indicated by the solid line through the blocks, with the above arrow indicating the direction of transcription. ( C ) CpG methylation profile at the endogenous host copy of ANKYRIN1 promoter. The region identical with ANKYRIN1 promoter sequence in the SB -IHK-DsRed is indicated in the schematic representation above the blocks of CpG methylation data derived from individual bisulfite-PCR amplicons. The cell clone from which genomic DNA was isolated is indicated at right, and methylated CpGs indicated by filled circles. The absence of CpG methylation at this region was also verified in the cell clone MS14 (data not shown).

    Techniques Used: DNA Methylation Assay, Combined Bisulfite Restriction Analysis Assay, CpG Methylation Assay, Polymerase Chain Reaction, Clone Assay, Methylation, Genomic Sequencing, Derivative Assay, Amplification, Blocking Assay, Sequencing, Isolation

    26) Product Images from "Identification of fur and fldA Homologs and a Pasteurella multocida tbpA Homolog in Histophilus ovis and Effects of Iron Availability on Their Transcription"

    Article Title: Identification of fur and fldA Homologs and a Pasteurella multocida tbpA Homolog in Histophilus ovis and Effects of Iron Availability on Their Transcription

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.184.9.2539-2542.2002

    Genetic organization of the tbpA genes in strains 9L and 3384Y. Putative −35, −10, and Shine-Dalgarno (SD) sequences are underlined, a potential Fur-binding site is in bold italics, start and stop codons are in bold, and inverted repeats, possibly involved in transcriptional termination, are in underlined italics.
    Figure Legend Snippet: Genetic organization of the tbpA genes in strains 9L and 3384Y. Putative −35, −10, and Shine-Dalgarno (SD) sequences are underlined, a potential Fur-binding site is in bold italics, start and stop codons are in bold, and inverted repeats, possibly involved in transcriptional termination, are in underlined italics.

    Techniques Used: Binding Assay

    27) Product Images from "CK1α overexpression correlates with poor survival in colorectal cancer"

    Article Title: CK1α overexpression correlates with poor survival in colorectal cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-018-4019-0

    Tumor differentiation-dependent investigation of CK1α RNA expression. a Box plot representing group comparison of relative CK1α RNA expression in low-grade and high-grade CRC patients. b Kaplan-Meier plot displaying the overall survival of Grade 3 and Grade 4 tumors of CRC patients, divided according to relative CK1α RNA expression. *** p
    Figure Legend Snippet: Tumor differentiation-dependent investigation of CK1α RNA expression. a Box plot representing group comparison of relative CK1α RNA expression in low-grade and high-grade CRC patients. b Kaplan-Meier plot displaying the overall survival of Grade 3 and Grade 4 tumors of CRC patients, divided according to relative CK1α RNA expression. *** p

    Techniques Used: RNA Expression

    28) 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

    29) Product Images from "Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan"

    Article Title: Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan

    Journal: Microbiology

    doi: 10.1099/mic.0.037507-0

    Enzymic analysis. Mannosyltransferase capping assay with synthetic Ara 6 as acceptor and membranes from M. marinum wild-type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.), and as control, membranes from M. marinum wild-type without acceptor. Shown are means of triplicates; error bars, sd .
    Figure Legend Snippet: Enzymic analysis. Mannosyltransferase capping assay with synthetic Ara 6 as acceptor and membranes from M. marinum wild-type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.), and as control, membranes from M. marinum wild-type without acceptor. Shown are means of triplicates; error bars, sd .

    Techniques Used: Acetylene Reduction Assay

    Physical appearance of liquid-grown M. marinum wild type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.). (a) OD 600 measurements during growth in 7H9+0.05 % Tween 80 with agitation. Shown are means of three independently grown cultures per strain; error bars sd . (b) M. marinum Δ MMAR_2380 shows increased aggregation as compared with the wild-type and the complemented strains (see Supplementary Fig. S4 for colour pictures).
    Figure Legend Snippet: Physical appearance of liquid-grown M. marinum wild type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.). (a) OD 600 measurements during growth in 7H9+0.05 % Tween 80 with agitation. Shown are means of three independently grown cultures per strain; error bars sd . (b) M. marinum Δ MMAR_2380 shows increased aggregation as compared with the wild-type and the complemented strains (see Supplementary Fig. S4 for colour pictures).

    Techniques Used:

    Absence of the mannose cap on mutant LAM. (a) Cell lysates from M. marinum wild-type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.) strains and M. smegmatis wild-type (control for ‘capless’ LAM) were immunoblotted with α -AraLAM antibody F30-5, which recognizes LAM, and α -ManLAM antibody 55.92.1A1, which recognizes the mannose cap. (b) Mannooligosaccharide cap analysis of LAM. Purified and partially degraded LAM was analysed for the presence of the mannose caps by CE. Shown are the profiles of LAM purified from M. marinum E11 (trace 1) and M. marinum Δ MMAR_2380 (trace 2). A, Ara-APTS; M, Man-APTS; S, internal standard, mannoheptose-APTS; AM, Manp- α (1→5)-Ara-APTS (monomannoside cap); AMM, Manp- α (1→2)-Manp- α (1→5)-Ara-APTS (dimannoside cap).
    Figure Legend Snippet: Absence of the mannose cap on mutant LAM. (a) Cell lysates from M. marinum wild-type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.) strains and M. smegmatis wild-type (control for ‘capless’ LAM) were immunoblotted with α -AraLAM antibody F30-5, which recognizes LAM, and α -ManLAM antibody 55.92.1A1, which recognizes the mannose cap. (b) Mannooligosaccharide cap analysis of LAM. Purified and partially degraded LAM was analysed for the presence of the mannose caps by CE. Shown are the profiles of LAM purified from M. marinum E11 (trace 1) and M. marinum Δ MMAR_2380 (trace 2). A, Ara-APTS; M, Man-APTS; S, internal standard, mannoheptose-APTS; AM, Manp- α (1→5)-Ara-APTS (monomannoside cap); AMM, Manp- α (1→2)-Manp- α (1→5)-Ara-APTS (dimannoside cap).

    Techniques Used: Mutagenesis, Laser Capture Microdissection, Purification, Acetylene Reduction Assay

    Reduced [1,2- 14 C]acetate incorporation in the polar lipids PIM 6 , LM and LAM from M. marinum Δ MMAR_2380 . (a) Extracted crude lipoglycans from acetate-labelled delipidated cells (25 μg) from M. marinum wild-type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.) were counted for the incorporation of [1,2- 14 C]acetate and analysed by SDS-PAGE/autoradiography. Shown is the average of two independent experiments. (b) [1,2- 14 C]Acetate-labelled M. marinum cultures were processed and polar lipids were applied (25 000 c.p.m.) to the corners of 6.6×6.6 cm pieces of aluminium-backed TLC plates and analysed using the 2D solvent system E as described in Methods. Plates were dried and autoradiograms were produced by overnight exposure of Kodak X-Omat AR film to the TLC plates to reveal [1,2- 14 C]acetate-labelled lipids. PI, non-mannosylated, diacylated phosphatidylinositol anchor; PIM 2 , phosphatidylinositol dimannoside; PIM 6 , phosphatidylinositol hexamannoside; Ac 1 PIM, tri-acylated PIM; Ac 2 PIM, tetra-acylated PIM. Unassigned spots in the figure are lipooligosaccharides, and in the upper-right corner are diphosphatidylglycerol, phosphatidylethanolamine and unknown phospholipids ( Burguière et al. , 2005 ). Two independently prepared lipid extracts per strain were tested.
    Figure Legend Snippet: Reduced [1,2- 14 C]acetate incorporation in the polar lipids PIM 6 , LM and LAM from M. marinum Δ MMAR_2380 . (a) Extracted crude lipoglycans from acetate-labelled delipidated cells (25 μg) from M. marinum wild-type, Δ MMAR_2380 and complemented Δ MMAR_2380 (Δ MMAR_2380 comp.) were counted for the incorporation of [1,2- 14 C]acetate and analysed by SDS-PAGE/autoradiography. Shown is the average of two independent experiments. (b) [1,2- 14 C]Acetate-labelled M. marinum cultures were processed and polar lipids were applied (25 000 c.p.m.) to the corners of 6.6×6.6 cm pieces of aluminium-backed TLC plates and analysed using the 2D solvent system E as described in Methods. Plates were dried and autoradiograms were produced by overnight exposure of Kodak X-Omat AR film to the TLC plates to reveal [1,2- 14 C]acetate-labelled lipids. PI, non-mannosylated, diacylated phosphatidylinositol anchor; PIM 2 , phosphatidylinositol dimannoside; PIM 6 , phosphatidylinositol hexamannoside; Ac 1 PIM, tri-acylated PIM; Ac 2 PIM, tetra-acylated PIM. Unassigned spots in the figure are lipooligosaccharides, and in the upper-right corner are diphosphatidylglycerol, phosphatidylethanolamine and unknown phospholipids ( Burguière et al. , 2005 ). Two independently prepared lipid extracts per strain were tested.

    Techniques Used: Laser Capture Microdissection, SDS Page, Autoradiography, Thin Layer Chromatography, Produced

    NMR analysis of the glyco part of LAM from M. marinum wild-type (pattern 1) and Δ MMAR_2380 (pattern 2). 2D 1 H- 13 C heteronuclear multiple quantum coherence spectroscopy (1H- 13 C HMQC) spectra of LAM in D 2 O at 313 K are shown with expanded regions ( δ 1 H, 4.80–5.35; δ 13 C, 99–113). The different resonances are labelled with the abbreviated name of the corresponding glycosyl units. See Supplementary Fig. S1 for structure of ManLAM.
    Figure Legend Snippet: NMR analysis of the glyco part of LAM from M. marinum wild-type (pattern 1) and Δ MMAR_2380 (pattern 2). 2D 1 H- 13 C heteronuclear multiple quantum coherence spectroscopy (1H- 13 C HMQC) spectra of LAM in D 2 O at 313 K are shown with expanded regions ( δ 1 H, 4.80–5.35; δ 13 C, 99–113). The different resonances are labelled with the abbreviated name of the corresponding glycosyl units. See Supplementary Fig. S1 for structure of ManLAM.

    Techniques Used: Nuclear Magnetic Resonance, Laser Capture Microdissection, Spectroscopy

    30) Product Images from "Comparative Properties and Functions of Type 2 and Type 4 Pigeon Cryptochromes"

    Article Title: Comparative Properties and Functions of Type 2 and Type 4 Pigeon Cryptochromes

    Journal: Cellular and molecular life sciences : CMLS

    doi: 10.1007/s00018-018-2920-y

    Phylogenetic analysis, tissue distribution and subcellular localization of ClCrys A. Phylogenetic analysis of animal Crys. Bootstrap probabilities are represented by values near the nodes. Several examples of animal Crys are included as Type 1 Crys, Type 2 Crys and Type 4 Crys. Names of species and accession numbers of sequences can be acquired from the Material and Method section. B. Expression of ClCry1, ClCry2, ClCry4 and β-actin RNA in the 10 indicated pigeon tissues detected by RT-PCR. C. Subcellular localization of ClCry1 and ClCry4 is revealed by expressing EGFP-ClCry fusion protein in cells. The five columns left to right show EGFP-Cry fusion protein (GFP-CRY), propidium iodide staining (PI), Merge 1 (Merging 1st and 2nd columns), bright field (BF), and Merge 2 (Merging 3rd and 4th columns). The top two rows illustrate GFP-ClCry1, and the bottom two rows illustrate GFP-ClCry4.
    Figure Legend Snippet: Phylogenetic analysis, tissue distribution and subcellular localization of ClCrys A. Phylogenetic analysis of animal Crys. Bootstrap probabilities are represented by values near the nodes. Several examples of animal Crys are included as Type 1 Crys, Type 2 Crys and Type 4 Crys. Names of species and accession numbers of sequences can be acquired from the Material and Method section. B. Expression of ClCry1, ClCry2, ClCry4 and β-actin RNA in the 10 indicated pigeon tissues detected by RT-PCR. C. Subcellular localization of ClCry1 and ClCry4 is revealed by expressing EGFP-ClCry fusion protein in cells. The five columns left to right show EGFP-Cry fusion protein (GFP-CRY), propidium iodide staining (PI), Merge 1 (Merging 1st and 2nd columns), bright field (BF), and Merge 2 (Merging 3rd and 4th columns). The top two rows illustrate GFP-ClCry1, and the bottom two rows illustrate GFP-ClCry4.

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

    31) Product Images from "Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors"

    Article Title: Site-Specific Chemoenzymatic Labeling of Aerolysin Enables the Identification of New Aerolysin Receptors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0109883

    Strategies for site-specific labeling of proaerolysin. A Structure of the proaerolysin monomer (PDB: 1PRE). Proaerolysin consists of several different domains, two of which are responsible for receptor binding (domains 1 and 2), one containsing the trans-membrane domain, and the C-terminal peptide (CP), which functions as a chaperone and dissociates from the rest of the complex upon heptamer association and pore formation. B Sortase reaction mechanism. C-terminal sortagging: sortase cleaves after threonine in the context of its recognition motif resulting in the formation of a new covalent bond with the N-terminus of an added oligoglycine or oligoalanine nucleophile coupled to a label of choice. N-terminal sortagging: the N-terminal glycine of proaerolysin is recognized as a nucleophile by sortase and conjugated to an LPXTG/A probe bearing a label. C Structures of probes used in this study. Not depicted is AAA.Alexa Fluor 647, which is similar to GGG.Alexa Fluor 647, but with alanine replacing glycine. PelB: periplasm targeting sequence, cleaved off by the producer bacteria upon export of proaerolysin to the periplasm. H6: hexahistidine handle for affinity purification. Protease cleavage sites are recognized by target cell surface proteases such as furin. CP: C-terminal peptide, serves as a chaperone for proaerolysin. Upon its loss, proaerolysin is converted to mature aerolysin (AeL). D Scheme for wild type (WT) and sortaggable versions of proaerolysin with their designations. The LPXTG/A pentapeptides are sortase recognition motifs.
    Figure Legend Snippet: Strategies for site-specific labeling of proaerolysin. A Structure of the proaerolysin monomer (PDB: 1PRE). Proaerolysin consists of several different domains, two of which are responsible for receptor binding (domains 1 and 2), one containsing the trans-membrane domain, and the C-terminal peptide (CP), which functions as a chaperone and dissociates from the rest of the complex upon heptamer association and pore formation. B Sortase reaction mechanism. C-terminal sortagging: sortase cleaves after threonine in the context of its recognition motif resulting in the formation of a new covalent bond with the N-terminus of an added oligoglycine or oligoalanine nucleophile coupled to a label of choice. N-terminal sortagging: the N-terminal glycine of proaerolysin is recognized as a nucleophile by sortase and conjugated to an LPXTG/A probe bearing a label. C Structures of probes used in this study. Not depicted is AAA.Alexa Fluor 647, which is similar to GGG.Alexa Fluor 647, but with alanine replacing glycine. PelB: periplasm targeting sequence, cleaved off by the producer bacteria upon export of proaerolysin to the periplasm. H6: hexahistidine handle for affinity purification. Protease cleavage sites are recognized by target cell surface proteases such as furin. CP: C-terminal peptide, serves as a chaperone for proaerolysin. Upon its loss, proaerolysin is converted to mature aerolysin (AeL). D Scheme for wild type (WT) and sortaggable versions of proaerolysin with their designations. The LPXTG/A pentapeptides are sortase recognition motifs.

    Techniques Used: Labeling, Binding Assay, Sequencing, Affinity Purification

    Identification of new aerolysin receptors. Biotin AeL.CP was used to identify new GPI-anchored proteins that bind Aerolysin . A Biotin.LPETG was attached to the N-terminus of proaerolysin via sortagging. The purified reaction product was analyzed by immunoblot. B HeLa cells were incubated with Biotin AeL.CP for 3 hours at 4°C and subsequently lysed with 0.5% NP-40. After pull-down with neutravidin beads, proteins were eluted, analyzed by SDS-PAGE, and subjected to mass spectrometry. Five GPI-anchored proteins were identified. UniProt accession codes are indicated. Peptides identified by mass spectrometry, lipidated amino acids, signal peptides, as well as peptides cleaved off from the pro-proteins are highlighted. C Binding of Biotin AeL.CP to mesothelin and to CD59 was verified by immunoblot.
    Figure Legend Snippet: Identification of new aerolysin receptors. Biotin AeL.CP was used to identify new GPI-anchored proteins that bind Aerolysin . A Biotin.LPETG was attached to the N-terminus of proaerolysin via sortagging. The purified reaction product was analyzed by immunoblot. B HeLa cells were incubated with Biotin AeL.CP for 3 hours at 4°C and subsequently lysed with 0.5% NP-40. After pull-down with neutravidin beads, proteins were eluted, analyzed by SDS-PAGE, and subjected to mass spectrometry. Five GPI-anchored proteins were identified. UniProt accession codes are indicated. Peptides identified by mass spectrometry, lipidated amino acids, signal peptides, as well as peptides cleaved off from the pro-proteins are highlighted. C Binding of Biotin AeL.CP to mesothelin and to CD59 was verified by immunoblot.

    Techniques Used: Purification, Incubation, SDS Page, Mass Spectrometry, Binding Assay

    Double-labeling of proaerolysin. Double-labeling was achieved with a two-step approach. A Schematic representation of the dual labeling strategy of proaerolysin. B We used SrtA Strep to install an oligoalanine coupled to the fluorophore AF647 at the C-terminus of proaerolysin, followed by a gel filtration purification step. C Elution profiles were analyzed by SDS-PAGE, fluorescence scan and coomassie stain. D The reaction product was subjected to the second round of sortagging with SrtA Staph7M and LPETG-coupled TAMRA fluorophore for N-terminal labeling. SrtA Staph does not recognize or cleave LPXTA, hence the C-terminal label remains intact. A single peak is observed on the elution profile as immobilized sortase was used for the reaction and removed prior to gel filtration. E Elution profiles were analyzed by SDS-PAGE followed by fluorescence scan.
    Figure Legend Snippet: Double-labeling of proaerolysin. Double-labeling was achieved with a two-step approach. A Schematic representation of the dual labeling strategy of proaerolysin. B We used SrtA Strep to install an oligoalanine coupled to the fluorophore AF647 at the C-terminus of proaerolysin, followed by a gel filtration purification step. C Elution profiles were analyzed by SDS-PAGE, fluorescence scan and coomassie stain. D The reaction product was subjected to the second round of sortagging with SrtA Staph7M and LPETG-coupled TAMRA fluorophore for N-terminal labeling. SrtA Staph does not recognize or cleave LPXTA, hence the C-terminal label remains intact. A single peak is observed on the elution profile as immobilized sortase was used for the reaction and removed prior to gel filtration. E Elution profiles were analyzed by SDS-PAGE followed by fluorescence scan.

    Techniques Used: Labeling, Filtration, Purification, SDS Page, Fluorescence, Staining

    Installation of a single label on proaerolysin. The fluorophore carboxytetramethylrhodamine (TAMRA) was installed at the N-terminus of aerolysin ( N AeL.CP), at the C-terminus of aerolysin upstream of the CP (AeL C ) and at the C-terminus of the C-terminal peptide (AeL.CP C ) with sortase. A, C, E Schematic representation of the sortagging reactions using of N AeL.CP, AeL.CP C, AeL C respectively. B, D Sortagging of N AeL.CP and AeL.CP C, respectively, with respective control conditions, resolved by SDS PAGE and imaged with a fluorescence scanner. Product is visible by fluorescent signal. SrtA Strep and SrtA Staph recognize and cleave LPXTA and LPXTG motives, respectively. F Purification of labeled AeL TAMRA , gel filtration. The first peak in the A280 elution profile corresponds to aerolysin, the second to sortase, and the third to free nucleophile. G Analysis of the first peak of the gel filtration elution profile with SDS PAGE followed by fluorescence image scan and Coomassie stain. A fraction of Ael C is not converted to fluorescent product.
    Figure Legend Snippet: Installation of a single label on proaerolysin. The fluorophore carboxytetramethylrhodamine (TAMRA) was installed at the N-terminus of aerolysin ( N AeL.CP), at the C-terminus of aerolysin upstream of the CP (AeL C ) and at the C-terminus of the C-terminal peptide (AeL.CP C ) with sortase. A, C, E Schematic representation of the sortagging reactions using of N AeL.CP, AeL.CP C, AeL C respectively. B, D Sortagging of N AeL.CP and AeL.CP C, respectively, with respective control conditions, resolved by SDS PAGE and imaged with a fluorescence scanner. Product is visible by fluorescent signal. SrtA Strep and SrtA Staph recognize and cleave LPXTA and LPXTG motives, respectively. F Purification of labeled AeL TAMRA , gel filtration. The first peak in the A280 elution profile corresponds to aerolysin, the second to sortase, and the third to free nucleophile. G Analysis of the first peak of the gel filtration elution profile with SDS PAGE followed by fluorescence image scan and Coomassie stain. A fraction of Ael C is not converted to fluorescent product.

    Techniques Used: SDS Page, Fluorescence, Purification, Labeling, Filtration, Staining

    32) Product Images from "Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites"

    Article Title: Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI29919

    Relative abundance of MCPs in MCP-1 –/– mice and newly created MCP-3 –/– , MCP-5 –/– , and MCP-2 –/– MCP-5 –/– mice.
    Figure Legend Snippet: Relative abundance of MCPs in MCP-1 –/– mice and newly created MCP-3 –/– , MCP-5 –/– , and MCP-2 –/– MCP-5 –/– mice.

    Techniques Used: Mouse Assay

    Monocyte retention in the bone marrow in MCP-1 –/– and MCP-3 –/– mice.
    Figure Legend Snippet: Monocyte retention in the bone marrow in MCP-1 –/– and MCP-3 –/– mice.

    Techniques Used: Mouse Assay

    Decrease in the level of 7/4 bri Ly-6G – monocytes in MCP-3 –/– and MCP-1 –/– mice.
    Figure Legend Snippet: Decrease in the level of 7/4 bri Ly-6G – monocytes in MCP-3 –/– and MCP-1 –/– mice.

    Techniques Used: Mouse Assay

    33) Product Images from "The Murine Gammaherpesvirus 68 ORF27 Gene Product Contributes to Intercellular Viral Spread"

    Article Title: The Murine Gammaherpesvirus 68 ORF27 Gene Product Contributes to Intercellular Viral Spread

    Journal:

    doi: 10.1128/JVI.79.8.5059-5068.2005

    The mature ORF27 gene product is a 48-kDa glycoprotein. (A) BHK-21 cells were infected with MHV-68 (2 PFU/cell), and 18 h later, were labeled with [ 35 S]cysteine-methionine for 1 h. ORF27 was then immunoprecipitated with the T6E11 or T8H3 MAb. Immunoprecipitations
    Figure Legend Snippet: The mature ORF27 gene product is a 48-kDa glycoprotein. (A) BHK-21 cells were infected with MHV-68 (2 PFU/cell), and 18 h later, were labeled with [ 35 S]cysteine-methionine for 1 h. ORF27 was then immunoprecipitated with the T6E11 or T8H3 MAb. Immunoprecipitations

    Techniques Used: Infection, Labeling, Immunoprecipitation

    Identification of the ORF27 gene product on virus-infected cell surfaces. (A) Aligning the MHV-68, KSHV, and EBV ORF27 homologs revealed differences in cytoplasmic tail length but similar arrangements of consensus N-linked glycan attachment sites and
    Figure Legend Snippet: Identification of the ORF27 gene product on virus-infected cell surfaces. (A) Aligning the MHV-68, KSHV, and EBV ORF27 homologs revealed differences in cytoplasmic tail length but similar arrangements of consensus N-linked glycan attachment sites and

    Techniques Used: Infection

    Disruption of ORF27 in the MHV-68 genome. (A) The sites of ORF27 mutagenesis are shown, as are relevant genomic and introduced restriction sites. Both mutations were located near the 5′ end of ORF27, downstream of the 3′ end of ORF26,
    Figure Legend Snippet: Disruption of ORF27 in the MHV-68 genome. (A) The sites of ORF27 mutagenesis are shown, as are relevant genomic and introduced restriction sites. Both mutations were located near the 5′ end of ORF27, downstream of the 3′ end of ORF26,

    Techniques Used: Mutagenesis

    34) Product Images from "Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus"

    Article Title: Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

    Journal: Parasites & Vectors

    doi: 10.1186/1756-3305-3-119

    Dynamic profiles of mRNA expression and enzyme activities of individual peptidases during the feeding of Ixodes ricinus females . For each time point, the guts were dissected from 15-20 females removed from four guinea pigs. Half of the guts were processed either for total RNA isolation or gut tissue extraction as described above. Gene expression profiles of the indicated enzymes were determined by qRT-PCR, using elongation factor 1 as a reference gene. The expressions were related to the maximum mRNA level set as 100%. Columns correspond to the average value of triplicate technical determinations, while error bars indicate the corresponding standard deviations. The enzyme activity curves of indicated peptidases were determined using specific fluorogenic substrates and shielding inhibitors to mask possible interference from other peptidases (see Table 1). Enzymatic activities in the homogenates were normalized per one gut tissue. The circles display the average activities obtained from triplicate measurements. The standard deviations were ≤5% of the average value and are not shown.
    Figure Legend Snippet: Dynamic profiles of mRNA expression and enzyme activities of individual peptidases during the feeding of Ixodes ricinus females . For each time point, the guts were dissected from 15-20 females removed from four guinea pigs. Half of the guts were processed either for total RNA isolation or gut tissue extraction as described above. Gene expression profiles of the indicated enzymes were determined by qRT-PCR, using elongation factor 1 as a reference gene. The expressions were related to the maximum mRNA level set as 100%. Columns correspond to the average value of triplicate technical determinations, while error bars indicate the corresponding standard deviations. The enzyme activity curves of indicated peptidases were determined using specific fluorogenic substrates and shielding inhibitors to mask possible interference from other peptidases (see Table 1). Enzymatic activities in the homogenates were normalized per one gut tissue. The circles display the average activities obtained from triplicate measurements. The standard deviations were ≤5% of the average value and are not shown.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Activity Assay

    35) Product Images from "The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]"

    Article Title: The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]

    Journal: The Plant Cell

    doi: 10.1105/tpc.110.080788

    MYC3 and MYC4 Interact with JAZ Repressors in Pull-Down Experiments.
    Figure Legend Snippet: MYC3 and MYC4 Interact with JAZ Repressors in Pull-Down Experiments.

    Techniques Used:

    Identification of MYC2, MYC3, and MYC4 DNA Binding Motifs in Vitro.
    Figure Legend Snippet: Identification of MYC2, MYC3, and MYC4 DNA Binding Motifs in Vitro.

    Techniques Used: Binding Assay, In Vitro

    MYC3 and MYC4 Interact with JAZ Repressors in Yeast Two-Hybrid Assays.
    Figure Legend Snippet: MYC3 and MYC4 Interact with JAZ Repressors in Yeast Two-Hybrid Assays.

    Techniques Used:

    Tissue Expression Patterns of MYC3 and MYC4 .
    Figure Legend Snippet: Tissue Expression Patterns of MYC3 and MYC4 .

    Techniques Used: Expressing

    36) Product Images from "The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]"

    Article Title: The Arabidopsis bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] bHLH Transcription Factors MYC3 and MYC4 Are Targets of JAZ Repressors and Act Additively with MYC2 in the Activation of Jasmonate Responses [C] [W]

    Journal: The Plant Cell

    doi: 10.1105/tpc.110.080788

    MYC3 and MYC4 Interact with JAZ Repressors in Pull-Down Experiments.
    Figure Legend Snippet: MYC3 and MYC4 Interact with JAZ Repressors in Pull-Down Experiments.

    Techniques Used:

    Identification of MYC2, MYC3, and MYC4 DNA Binding Motifs in Vitro.
    Figure Legend Snippet: Identification of MYC2, MYC3, and MYC4 DNA Binding Motifs in Vitro.

    Techniques Used: Binding Assay, In Vitro

    MYC3 and MYC4 Interact with JAZ Repressors in Yeast Two-Hybrid Assays.
    Figure Legend Snippet: MYC3 and MYC4 Interact with JAZ Repressors in Yeast Two-Hybrid Assays.

    Techniques Used:

    Tissue Expression Patterns of MYC3 and MYC4 .
    Figure Legend Snippet: Tissue Expression Patterns of MYC3 and MYC4 .

    Techniques Used: Expressing

    37) 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

    38) Product Images from "Mutant IDH1 Confers an in Vivo Growth in a Melanoma Cell Line with BRAF Mutation"

    Article Title: Mutant IDH1 Confers an in Vivo Growth in a Melanoma Cell Line with BRAF Mutation

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2010.12.011

    IDH1 and IDH2 mutations in melanoma. Amino acid alignments of IDH1 and IDH2 proteins among human, mouse, rat, and chicken homologues are shown in the top panels . Sequence chromatographies of the IDH1 and IDH2 genes in primary melanomas (T) and corresponding
    Figure Legend Snippet: IDH1 and IDH2 mutations in melanoma. Amino acid alignments of IDH1 and IDH2 proteins among human, mouse, rat, and chicken homologues are shown in the top panels . Sequence chromatographies of the IDH1 and IDH2 genes in primary melanomas (T) and corresponding

    Techniques Used: Sequencing

    39) 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

    40) Product Images from "Investigation of the Staphylococcus aureus GraSR Regulon Reveals Novel Links to Virulence, Stress Response and Cell Wall Signal Transduction Pathways"

    Article Title: Investigation of the Staphylococcus aureus GraSR Regulon Reveals Novel Links to Virulence, Stress Response and Cell Wall Signal Transduction Pathways

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0021323

    Identification of potential GraR-binding sites in the promoters of known GraR-regulated genes. (A) Primer extension analysis of mprF , dltXABCD and vraFG transcripts was carried out using total RNA extracted from S. aureus strain HG001 treated with 200 µg ml −1 colistin during mid-exponential growth at 37°C in TSB, using specific oligonucleotides for mprF , dltX and vraF (lanes 1 to 3 respectively). The corresponding Sanger dideoxy chain termination sequencing reactions (GATC) were carried out on PCR-generated DNA fragments corresponding to the respective upstream regions (see Table 5 ). The transcriptional start sites are boxed. (B) Alignment of the potential GraR DNA-binding sites in the mprF , dltXABCD and vraFG promoter regions. The potential GraR-binding site is shaded and conserved nucleotides are shown in white. Potential −35 and −10 sequences are underlined and the transcriptional start sites are indicated in bold.
    Figure Legend Snippet: Identification of potential GraR-binding sites in the promoters of known GraR-regulated genes. (A) Primer extension analysis of mprF , dltXABCD and vraFG transcripts was carried out using total RNA extracted from S. aureus strain HG001 treated with 200 µg ml −1 colistin during mid-exponential growth at 37°C in TSB, using specific oligonucleotides for mprF , dltX and vraF (lanes 1 to 3 respectively). The corresponding Sanger dideoxy chain termination sequencing reactions (GATC) were carried out on PCR-generated DNA fragments corresponding to the respective upstream regions (see Table 5 ). The transcriptional start sites are boxed. (B) Alignment of the potential GraR DNA-binding sites in the mprF , dltXABCD and vraFG promoter regions. The potential GraR-binding site is shaded and conserved nucleotides are shown in white. Potential −35 and −10 sequences are underlined and the transcriptional start sites are indicated in bold.

    Techniques Used: Binding Assay, Sequencing, Polymerase Chain Reaction, Generated

    The graXRS operon is transcribed from a σ A promoter. (A) The graXRS / vraFG locus of S . aureus HG001. (B) Primer extension analysis of graXRS mRNA was carried out using total RNA extracted from S. aureus strain HG001 during mid-exponential growth in TSB at 37°C. Primer extension experiments were performed using the graX -specific oligonucleotide MF63 (lane 1). The corresponding Sanger dideoxy chain termination sequencing reactions (GATC) were carried out on a PCR-generated DNA fragment fragment corresponding to the graX upstream region (MF62/MF63). The transcriptional start site is boxed. (C) Nucleotide sequence of the graXRS operon upstream region. Potential σ A -type -35 and −10 sequences are boxed and the transcriptional start site is labelled +1.
    Figure Legend Snippet: The graXRS operon is transcribed from a σ A promoter. (A) The graXRS / vraFG locus of S . aureus HG001. (B) Primer extension analysis of graXRS mRNA was carried out using total RNA extracted from S. aureus strain HG001 during mid-exponential growth in TSB at 37°C. Primer extension experiments were performed using the graX -specific oligonucleotide MF63 (lane 1). The corresponding Sanger dideoxy chain termination sequencing reactions (GATC) were carried out on a PCR-generated DNA fragment fragment corresponding to the graX upstream region (MF62/MF63). The transcriptional start site is boxed. (C) Nucleotide sequence of the graXRS operon upstream region. Potential σ A -type -35 and −10 sequences are boxed and the transcriptional start site is labelled +1.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Generated

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    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). ..

    Transfection:

    Article Title: Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact
    Article Snippet: .. Extraction of DNA and RNA from Cells DNA and RNA were extracted from transfected and non-transfected hepatoma cells in a Magnapure robot (Roche Applied Science, Germany) using the Total NA protocol. .. Harvested cells were washed in 1 mL PBS and after centrifugation at 5000 rpm for 3 min the pellet was re-suspended in 800 µL RLT lysis buffer (Qiagen Sciences, MD, USA) before extraction.

    Amplification:

    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
    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). ..

    SYBR Green Assay:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    Polymerase Chain Reaction:

    Article Title: The invasiveness of human cervical cancer associated to the function of NaV1.6 channels is mediated by MMP-2 activity
    Article Snippet: .. The first strand cDNA product (250 ng) was used as template in 10 µl PCR reactions with final concentrations of 200 µM dNTP, 0.3 µM primers for SCN8A Exon 18 (forward primer 5′-AAGTGGACAGCCTATGGCTTCG-3′, reverse primer 5′-TGTTGACATCTTCAATTTCAAATCGG-3′), 1.5 mM MgCl2 , and 1.3 U of enzyme mix (Expand High Fidelity PCR System, Roche Diagnostics; Mannheim, Germany). ..

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    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
    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.

    Reverse Transcription Polymerase Chain Reaction:

    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).

    Chromatin Immunoprecipitation:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

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  • 91
    Roche high fidelity pcr kit
    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in <t>Igκ</t> wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative <t>PCR</t> via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.
    High Fidelity Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 91/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche expand high fidelity pcr system kit
    <t>PCR</t> amplification and cloning of <t>DNA</t> from experimental tissues
    Expand High Fidelity Pcr System Kit, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.

    Journal: Frontiers in Immunology

    Article Title: A Novel Pax5-Binding Regulatory Element in the Ig? Locus

    doi: 10.3389/fimmu.2014.00240

    Figure Lengend Snippet: Pax5-binding within the Dm element . (A) Comparison of the putative Pax5-binding site within the Dm element, known Pax5-binding site in sea urchin H2a-2.2 promoter and Pax5 consensus sequence. Bases which match the consensus sequence are marked in red, while bases which do not match are marked in gray. (B) Electro-mobility shift assay (EMSA) of Taq I– Bst EII 70 bp fragment from the Dm element, containing the putative Pax5-binding site, with the indicated hematopoietic cell type nuclear extracts. Red arrowhead indicates B cell lineage-specific shift. (C) Competition EMSA. Unlabeled H2a-2.2 probe competes with radioactive Taq I– Bst EII Dm probe and reduces shift in B lineage cell extracts (M12, 70Z/3), and in extracts of fibroblast cells (cop8) transfected with a Pax5 expression vector. Nonsense unlabeled probe (ns) is unable to compete with Dm probe. Red arrowhead indicates specific shift. (D) Supershift assay with Taq I– Bst EII Dm probe, which was incubated with antibodies (ab) raised against the DNA-binding site of Pax5 (A1), the transactivation domain (A2), or general rabbit antisera (P.I.). (E) DNase I footprinting assay on end-labeled Taq I– Sac II probe from the Dm element. Labeled probe was incubated with nuclear extracts from the indicated cell types. A control Maxam and Gilbert reaction (G/A) was run in parallel. Footprint specific to Pax5-expressing M12 cells is indicated with a red arrow. Location of the putative Pax5-binding site is marked in red on the nucleotide sequence. (F) Chromatin immunoprecipitation (ChIP) enrichment of Pax5 at the Dm element in Igκ wt/ΔDm cultured pre-B cells. Enrichment was measured by semi-quantitative PCR via comparison of the input DNA (Input) to the immunoprecipitated fraction (αPax5), using primers specific to the Dm element (Dm) and the deleted allele (ΔDm). One and three times the amount of PCR template were run in parallel to ensure linearity. Positive (CD19 promoter) and negative (β-actin promoter) controls for Pax5-binding were analyzed in parallel to ensure specificity of the enrichment. ChIP with a non-specific antibody (IgG) does not enrich the Dm element.

    Article Snippet: WT and ΔDm rearranged Igκ alleles were amplified with Vκ-Degenerate 5′-GTCCCTGCCAGGTTYAGTGGCAGTGGRTCWRGGAC-3′ and R3-1 5′-CAGACCCTGGTCTAATGGTTTGTAACCACATGGG-3′ primers using high fidelity PCR kit (Roche) with an initial denaturation of 4 min at 94°C, followed by 35 cycles of denaturation at 94°C for 15 s and annealing combined with elongation at 68°C for 2 min. 3′ A-overhang nucleotides were added by 20 min incubation with Taq polymerase and ATP at 72°C.

    Techniques: Binding Assay, Sequencing, Electro Mobility Shift Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Footprinting, Labeling, Chromatin Immunoprecipitation, Cell Culture, Real-time Polymerase Chain Reaction, Immunoprecipitation, Polymerase Chain Reaction

    Identification of C. sordellii and C. perfringens in confirmed CTS cases by the microsphere assay. Cases 1–10: C. sordellii cases confirmed by conventional PCR and sequencing. Cases 11–18: C. perfringens cases confirmed by conventional PCR and sequencing. No cross-reactivity was noted.

    Journal: Infectious Diseases in Obstetrics and Gynecology

    Article Title: Rapid, Simultaneous Detection of Clostridium sordellii and Clostridium perfringens in Archived Tissues by a Novel PCR-Based Microsphere Assay: Diagnostic Implications for Pregnancy-Associated Toxic Shock Syndrome Cases

    doi: 10.1155/2012/972845

    Figure Lengend Snippet: Identification of C. sordellii and C. perfringens in confirmed CTS cases by the microsphere assay. Cases 1–10: C. sordellii cases confirmed by conventional PCR and sequencing. Cases 11–18: C. perfringens cases confirmed by conventional PCR and sequencing. No cross-reactivity was noted.

    Article Snippet: Initially, phospholipase C genes of C. sordellii and C. perfringens were amplified using a High Fidelity PCR Kit (Roche), 0.3 μ M of each primer (final concentration), and 5 μ L of template DNA sample in a 50 μ L final reaction volume, following the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing

    PCR amplification and cloning of DNA from experimental tissues

    Journal: AIDS Research and Human Retroviruses

    Article Title: Reduced Genetic Diversity in Lymphoid and Central Nervous System Tissues and Selection-Induced Tissue-Specific Compartmentalization of Neuropathogenic SIVsmmFGb during Acute Infection

    doi: 10.1089/aid.2008.0240

    Figure Lengend Snippet: PCR amplification and cloning of DNA from experimental tissues

    Article Snippet: The nef, env , and int genes were amplified from proviral DNA by nested PCR, using an Expand High Fidelity PCR system kit (Roche, Indianapolis, IN), according to the manufacturer's protocol.

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay

    SIVsmmFGb stock virus sequence group composition. As described in and Materials and Methods, 16 SIVsmmFGb stock virus templates, with different RNA and DNA dilutions, were used for nested PCR reactions of the env V1 region, nef , and int . At least

    Journal: AIDS Research and Human Retroviruses

    Article Title: Reduced Genetic Diversity in Lymphoid and Central Nervous System Tissues and Selection-Induced Tissue-Specific Compartmentalization of Neuropathogenic SIVsmmFGb during Acute Infection

    doi: 10.1089/aid.2008.0240

    Figure Lengend Snippet: SIVsmmFGb stock virus sequence group composition. As described in and Materials and Methods, 16 SIVsmmFGb stock virus templates, with different RNA and DNA dilutions, were used for nested PCR reactions of the env V1 region, nef , and int . At least

    Article Snippet: The nef, env , and int genes were amplified from proviral DNA by nested PCR, using an Expand High Fidelity PCR system kit (Roche, Indianapolis, IN), according to the manufacturer's protocol.

    Techniques: Sequencing, Nested PCR

    Characterization of the FirS iron-binding motif. The induction of ygiW (A) and firR (B) in response to Fe 2+ was measured by qRT-PCR. The ability of wild-type 2019, the Δ firS mutant KK009, KHS1 ( firS Y149G, R150T), KHS3 ( firS D148A), KHS4 ( firS E151G, D152S), and KHS5 ( firS + ) to respond to Fe 2+ was tested. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a Ferrous Iron-Responsive Two-Component System in Nontypeable Haemophilus influenzae

    doi: 10.1128/JB.01465-12

    Figure Lengend Snippet: Characterization of the FirS iron-binding motif. The induction of ygiW (A) and firR (B) in response to Fe 2+ was measured by qRT-PCR. The ability of wild-type 2019, the Δ firS mutant KK009, KHS1 ( firS Y149G, R150T), KHS3 ( firS D148A), KHS4 ( firS E151G, D152S), and KHS5 ( firS + ) to respond to Fe 2+ was tested. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Article Snippet: For probe synthesis, DNA fragments internal to each gene were amplified by PCR using primers 1709F8 and 1709R7 ( ygiW ) and 1708F3 and 1708R3 ( firR ) and the Expand high-fidelity PCR kit (Roche).

    Techniques: Binding Assay, Quantitative RT-PCR, Mutagenesis, Expressing

    Thermoresponsive induction of ygiW (A) and firR (B). Cultures of wild-type NTHI 2019, the Δ firR mutant (NB004), the Δ firS mutant (KK009), the complemented firR mutant ( firR + ; KHS2), and the complemented firS mutant ( firS + ; KHS5) were grown in sRPMI at 37°C to early log phase and shifted to 9°C for 30 min prior to RNA extraction. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene when cultures were incubated at 37°C. The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a Ferrous Iron-Responsive Two-Component System in Nontypeable Haemophilus influenzae

    doi: 10.1128/JB.01465-12

    Figure Lengend Snippet: Thermoresponsive induction of ygiW (A) and firR (B). Cultures of wild-type NTHI 2019, the Δ firR mutant (NB004), the Δ firS mutant (KK009), the complemented firR mutant ( firR + ; KHS2), and the complemented firS mutant ( firS + ; KHS5) were grown in sRPMI at 37°C to early log phase and shifted to 9°C for 30 min prior to RNA extraction. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene when cultures were incubated at 37°C. The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Article Snippet: For probe synthesis, DNA fragments internal to each gene were amplified by PCR using primers 1709F8 and 1709R7 ( ygiW ) and 1708F3 and 1708R3 ( firR ) and the Expand high-fidelity PCR kit (Roche).

    Techniques: Mutagenesis, RNA Extraction, Expressing, Quantitative RT-PCR, Incubation

    Characterization of firR mutants in Fe 2+ -responsive induction of ygiW . The expression of ygiW in wild-type 2019, NB004 (Δ firR ), JWJ154 ( firR D51A), and KHS2 ( firR + ) was measured by qRT-PCR. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Journal: Journal of Bacteriology

    Article Title: Characterization of a Ferrous Iron-Responsive Two-Component System in Nontypeable Haemophilus influenzae

    doi: 10.1128/JB.01465-12

    Figure Lengend Snippet: Characterization of firR mutants in Fe 2+ -responsive induction of ygiW . The expression of ygiW in wild-type 2019, NB004 (Δ firR ), JWJ154 ( firR D51A), and KHS2 ( firR + ) was measured by qRT-PCR. Expression of each gene was measured by qRT-PCR and compared to the expression of each gene in cultures grown without the addition of exogenous FeCl 2 . The data presented are means and standard deviations from two experiments, each performed in triplicate.

    Article Snippet: For probe synthesis, DNA fragments internal to each gene were amplified by PCR using primers 1709F8 and 1709R7 ( ygiW ) and 1708F3 and 1708R3 ( firR ) and the Expand high-fidelity PCR kit (Roche).

    Techniques: Expressing, Quantitative RT-PCR