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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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    Images

    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 "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

    4) Product Images from "A Novel Role for the Transcription Factor Cwt1p as a Negative Regulator of Nitrosative Stress in Candida albicans"

    Article Title: A Novel Role for the Transcription Factor Cwt1p as a Negative Regulator of Nitrosative Stress in Candida albicans

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043956

    Cwt1p is a direct regulator of nitrosative stress responsive-genes. Relationship between Cwt1p-bound genes and genes showing altered expression in the cwt1 mutant challenged by 0.1 mM DPTA NONOate during 15 min. The symbol (*) and (**) indicate a significant difference compared to the WT strain using t -test (P
    Figure Legend Snippet: Cwt1p is a direct regulator of nitrosative stress responsive-genes. Relationship between Cwt1p-bound genes and genes showing altered expression in the cwt1 mutant challenged by 0.1 mM DPTA NONOate during 15 min. The symbol (*) and (**) indicate a significant difference compared to the WT strain using t -test (P

    Techniques Used: Expressing, Mutagenesis

    Cwt1p bound the promoter region of the nitric oxide dioxygenase YHB1 and controls negatively its transcription. ( A ) Cwt1p is detected at the YHB1 promoter using tiled ChIP-qPCR, and the enrichment signal overlaps precisely its DNA-binding motif. Cwt1p occupancy was assessed in the presence and the absence of nitrosative stress. The nitric oxide-responsive element (NORE) recognized by the TF Cta4p is also shown. ( B ) Average expression of the nitric oxide dioxygenase YHB1 in response to 0.1 mM DPTA NONOate is shown in the wt and cwt1 strains in two independent biological replicates. Fold changes were estimated by using the coding sequence of the C. albicans ACT1 ORF as a reference. Fold enrichments of the tested coding sequences were estimated using the comparative ΔΔCt method.
    Figure Legend Snippet: Cwt1p bound the promoter region of the nitric oxide dioxygenase YHB1 and controls negatively its transcription. ( A ) Cwt1p is detected at the YHB1 promoter using tiled ChIP-qPCR, and the enrichment signal overlaps precisely its DNA-binding motif. Cwt1p occupancy was assessed in the presence and the absence of nitrosative stress. The nitric oxide-responsive element (NORE) recognized by the TF Cta4p is also shown. ( B ) Average expression of the nitric oxide dioxygenase YHB1 in response to 0.1 mM DPTA NONOate is shown in the wt and cwt1 strains in two independent biological replicates. Fold changes were estimated by using the coding sequence of the C. albicans ACT1 ORF as a reference. Fold enrichments of the tested coding sequences were estimated using the comparative ΔΔCt method.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Expressing, Sequencing

    Growth inhibition assay by the nitric oxide donor DPTA NONOate. ( A ) Growth inhibition assay of selected mutant strains exposed to 0.3 and 1 mM DPTA NONOate during 3.5 hours. Growth inhibition rate was normalized relative to the 0 mM DPTA NONOate condition for each strain. Results are from three experiments. ( B ) Growth defect of cwt1 and cta4 strains was confirmed by an independent NO inhibition assay using different DPTA NONOate concentrations: 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 1 mM. Growth of the wt (SN152) and cwt1 / cwt1 /CWT1 strains is also shown. Values are average of three replicates. Error bars are standard deviations of triplicates. The symbol (*) and (**) indicate a significant difference compared to the WT strain using t -test (P
    Figure Legend Snippet: Growth inhibition assay by the nitric oxide donor DPTA NONOate. ( A ) Growth inhibition assay of selected mutant strains exposed to 0.3 and 1 mM DPTA NONOate during 3.5 hours. Growth inhibition rate was normalized relative to the 0 mM DPTA NONOate condition for each strain. Results are from three experiments. ( B ) Growth defect of cwt1 and cta4 strains was confirmed by an independent NO inhibition assay using different DPTA NONOate concentrations: 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 1 mM. Growth of the wt (SN152) and cwt1 / cwt1 /CWT1 strains is also shown. Values are average of three replicates. Error bars are standard deviations of triplicates. The symbol (*) and (**) indicate a significant difference compared to the WT strain using t -test (P

    Techniques Used: Growth Inhibition Assay, Mutagenesis, Inhibition

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

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

    Journal: PLoS Medicine

    doi: 10.1371/journal.pmed.0030443

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

    Techniques Used: Labeling

    6) Product Images from "Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization"

    Article Title: Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600565

    BET3 dimer in the crystal and in solution. ( A ) Cα trace of the BET3 dimer viewed down the crystallographic two-fold axis. Secondary structural elements interacting at the dimer interface are labeled. The two subunits are shown in different colors.
    Figure Legend Snippet: BET3 dimer in the crystal and in solution. ( A ) Cα trace of the BET3 dimer viewed down the crystallographic two-fold axis. Secondary structural elements interacting at the dimer interface are labeled. The two subunits are shown in different colors.

    Techniques Used: Labeling

    Stereo diagrams of human BET3. ( A ) Schematic representation of the BET3 monomer with the helices and strands colored red and cyan, respectively, and labeled. The palmitate molecule covalently linked to residue C68 through a thioester bond is shown in
    Figure Legend Snippet: Stereo diagrams of human BET3. ( A ) Schematic representation of the BET3 monomer with the helices and strands colored red and cyan, respectively, and labeled. The palmitate molecule covalently linked to residue C68 through a thioester bond is shown in

    Techniques Used: Labeling

    ( A ) Membrane preparations from yeast expressing BET3 and BET3 C68S. Cleared lysates were subjected to ultracentrifugation. Total cell lysate (T), cytosolic (C), and membrane (M, 10-fold concentrated) fractions were analyzed by SDS–PAGE and immunoblotting.
    Figure Legend Snippet: ( A ) Membrane preparations from yeast expressing BET3 and BET3 C68S. Cleared lysates were subjected to ultracentrifugation. Total cell lysate (T), cytosolic (C), and membrane (M, 10-fold concentrated) fractions were analyzed by SDS–PAGE and immunoblotting.

    Techniques Used: Expressing, SDS Page

    Covalent modification of BET3 with palmitate. ( A ) A portion of the (2 m ∣ F obs ∣− D ∣ F calc ∣)α calc
    Figure Legend Snippet: Covalent modification of BET3 with palmitate. ( A ) A portion of the (2 m ∣ F obs ∣− D ∣ F calc ∣)α calc

    Techniques Used: Modification

    7) Product Images from "Genetic Dissection of the Regulatory Network Associated with High c-di-GMP Levels in Pseudomonas putida KT2440"

    Article Title: Genetic Dissection of the Regulatory Network Associated with High c-di-GMP Levels in Pseudomonas putida KT2440

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01093

    Alignment of CfcR and CfcD REC domains. Domains were defined as described in the MIST database ( Ulrich and Zhulin, 2010 ) and aligned using EMBOSS Needle ( Rice et al., 2000 ). Phosphorylated D65 in CfcR is highlighted in gray.
    Figure Legend Snippet: Alignment of CfcR and CfcD REC domains. Domains were defined as described in the MIST database ( Ulrich and Zhulin, 2010 ) and aligned using EMBOSS Needle ( Rice et al., 2000 ). Phosphorylated D65 in CfcR is highlighted in gray.

    Techniques Used:

    8) Product Images from "Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs ▿ mRNAs ▿ †"

    Article Title: Both Linear and Discontinuous Ribosome Scanning Are Used for Translation Initiation from Bicistronic Human Immunodeficiency Virus Type 1 env mRNAs ▿ mRNAs ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01028-06

    Increasing the strength of or removing the Rev AUG modulates Vpu expression. (A) Mutations in the env2 cDNA constructs that either strengthened the Rev AUG codon (underlined; RevK) or removed it (Rev − ). (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pEGFP, and pDM128 without (B) or with (C) pLTR rev1 . Rev activity was measured by CAT assay. Env and Vpu were detected by immunoblotting followed by densitometry analysis, and the percentage of expression relative to the expression of the env1 R38 mutant (E1R38; set at 100%) is graphed. wt, wild type; Mk, mock (no env or pLTR rev1 ); E1 and E2, pDR env1 and pDR env2 ; E2K, env2 cDNA construct expressing RevK mutation; E2R38, env2 cDNA construct expressing R38 mutation; E2KR38, env2 cDNA construct expressing RevK and R38 mutations; E2R−, env2 cDNA construct expressing Rev − mutation.
    Figure Legend Snippet: Increasing the strength of or removing the Rev AUG modulates Vpu expression. (A) Mutations in the env2 cDNA constructs that either strengthened the Rev AUG codon (underlined; RevK) or removed it (Rev − ). (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pEGFP, and pDM128 without (B) or with (C) pLTR rev1 . Rev activity was measured by CAT assay. Env and Vpu were detected by immunoblotting followed by densitometry analysis, and the percentage of expression relative to the expression of the env1 R38 mutant (E1R38; set at 100%) is graphed. wt, wild type; Mk, mock (no env or pLTR rev1 ); E1 and E2, pDR env1 and pDR env2 ; E2K, env2 cDNA construct expressing RevK mutation; E2R38, env2 cDNA construct expressing R38 mutation; E2KR38, env2 cDNA construct expressing RevK and R38 mutations; E2R−, env2 cDNA construct expressing Rev − mutation.

    Techniques Used: Expressing, Construct, Transfection, Activity Assay, Mutagenesis

    Adding an upstream, strong Kozak AUG codon in exon 2 of the env isoforms does not diminish Env expression. (A) Mutations in exon 2 of the env cDNA constructs that introduced either a strong Kozak AUG codon (KAUG) or the same mutations minus the AUG codon (KAGG). A UAA translation termination motif was included at codon 4 to avoid aberrant protein synthesis. (B) HeLa cells transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP were analyzed for Env and Vpu by immunoblotting and densitometry. The percentage of expression relative to that of the env5 construct (E5; set at 100%) is graphed. wt, wild type; E6, pDR env6 ; E5KAGG and E6KAGG, env5 and env6 constructs without the Kozak AUG codon; E5KAUG and E6KAUG, env5 and env6 constructs with the Kozak AUG codon.
    Figure Legend Snippet: Adding an upstream, strong Kozak AUG codon in exon 2 of the env isoforms does not diminish Env expression. (A) Mutations in exon 2 of the env cDNA constructs that introduced either a strong Kozak AUG codon (KAUG) or the same mutations minus the AUG codon (KAGG). A UAA translation termination motif was included at codon 4 to avoid aberrant protein synthesis. (B) HeLa cells transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP were analyzed for Env and Vpu by immunoblotting and densitometry. The percentage of expression relative to that of the env5 construct (E5; set at 100%) is graphed. wt, wild type; E6, pDR env6 ; E5KAGG and E6KAGG, env5 and env6 constructs without the Kozak AUG codon; E5KAUG and E6KAUG, env5 and env6 constructs with the Kozak AUG codon.

    Techniques Used: Expressing, Construct, Transfection

    Removing upstream AUG codons and changing Vpu ORF length does not alter consistent Env expression. (A) Mutations in the env cDNA constructs that removed the upstream Rev and/or Vpu AUG codon (underlined; Rev− and/or Vpu−) or shortened the Vpu ORF at the position corresponding to amino acid 2 (Vpu2) or 35 (ΔU35). Numbers indicate amino acid positions corresponding to the codons. (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP. Env and Vpu expression was analyzed by immunoblotting and then densitometry. The percentage of expression relative to that of the env1 R38 mutant (E1R; set at 100%) is graphed for the env isoforms lacking upstream AUG codons (B) or containing altered Vpu ORF lengths (C). wt, wild type; Mk, mock (no env ); E1R to E3R, pDR env1 to pDR env3 cDNA constructs containing the Rev R38 premature termination codon mutation; E2−, env2 cDNA construct expressing the Rev − mutation; E2V−, env2 cDNA construct expressing the Vpu − mutation; E2R−V−, env2 cDNA construct expressing the Rev − and Vpu − mutations; E1RΔU35 to E3RΔU35, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 35; E1RVpu2 to E3RVpu2, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 2.
    Figure Legend Snippet: Removing upstream AUG codons and changing Vpu ORF length does not alter consistent Env expression. (A) Mutations in the env cDNA constructs that removed the upstream Rev and/or Vpu AUG codon (underlined; Rev− and/or Vpu−) or shortened the Vpu ORF at the position corresponding to amino acid 2 (Vpu2) or 35 (ΔU35). Numbers indicate amino acid positions corresponding to the codons. (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP. Env and Vpu expression was analyzed by immunoblotting and then densitometry. The percentage of expression relative to that of the env1 R38 mutant (E1R; set at 100%) is graphed for the env isoforms lacking upstream AUG codons (B) or containing altered Vpu ORF lengths (C). wt, wild type; Mk, mock (no env ); E1R to E3R, pDR env1 to pDR env3 cDNA constructs containing the Rev R38 premature termination codon mutation; E2−, env2 cDNA construct expressing the Rev − mutation; E2V−, env2 cDNA construct expressing the Vpu − mutation; E2R−V−, env2 cDNA construct expressing the Rev − and Vpu − mutations; E1RΔU35 to E3RΔU35, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 35; E1RVpu2 to E3RVpu2, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 2.

    Techniques Used: Expressing, Construct, Transfection, Mutagenesis

    Increasing the strength of or removing the Rev AUG modulates Vpu expression. (A) Mutations in the env2 cDNA constructs that either strengthened the Rev AUG codon (underlined; RevK) or removed it (Rev − ). (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pEGFP, and pDM128 without (B) or with (C) pLTR rev1 . Rev activity was measured by CAT assay. Env and Vpu were detected by immunoblotting followed by densitometry analysis, and the percentage of expression relative to the expression of the env1 R38 mutant (E1R38; set at 100%) is graphed. wt, wild type; Mk, mock (no env or pLTR rev1 ); E1 and E2, pDR env1 and pDR env2 ; E2K, env2 cDNA construct expressing RevK mutation; E2R38, env2 cDNA construct expressing R38 mutation; E2KR38, env2 cDNA construct expressing RevK and R38 mutations; E2R−, env2 cDNA construct expressing Rev − mutation.
    Figure Legend Snippet: Increasing the strength of or removing the Rev AUG modulates Vpu expression. (A) Mutations in the env2 cDNA constructs that either strengthened the Rev AUG codon (underlined; RevK) or removed it (Rev − ). (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pEGFP, and pDM128 without (B) or with (C) pLTR rev1 . Rev activity was measured by CAT assay. Env and Vpu were detected by immunoblotting followed by densitometry analysis, and the percentage of expression relative to the expression of the env1 R38 mutant (E1R38; set at 100%) is graphed. wt, wild type; Mk, mock (no env or pLTR rev1 ); E1 and E2, pDR env1 and pDR env2 ; E2K, env2 cDNA construct expressing RevK mutation; E2R38, env2 cDNA construct expressing R38 mutation; E2KR38, env2 cDNA construct expressing RevK and R38 mutations; E2R−, env2 cDNA construct expressing Rev − mutation.

    Techniques Used: Expressing, Construct, Transfection, Activity Assay, Mutagenesis

    Removing upstream AUG codons and changing Vpu ORF length does not alter consistent Env expression. (A) Mutations in the env cDNA constructs that removed the upstream Rev and/or Vpu AUG codon (underlined; Rev− and/or Vpu−) or shortened the Vpu ORF at the position corresponding to amino acid 2 (Vpu2) or 35 (ΔU35). Numbers indicate amino acid positions corresponding to the codons. (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP. Env and Vpu expression was analyzed by immunoblotting and then densitometry. The percentage of expression relative to that of the env1 R38 mutant (E1R; set at 100%) is graphed for the env isoforms lacking upstream AUG codons (B) or containing altered Vpu ORF lengths (C). wt, wild type; Mk, mock (no env ); E1R to E3R, pDR env1 to pDR env3 cDNA constructs containing the Rev R38 premature termination codon mutation; E2−, env2 cDNA construct expressing the Rev − mutation; E2V−, env2 cDNA construct expressing the Vpu − mutation; E2R−V−, env2 cDNA construct expressing the Rev − and Vpu − mutations; E1RΔU35 to E3RΔU35, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 35; E1RVpu2 to E3RVpu2, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 2.
    Figure Legend Snippet: Removing upstream AUG codons and changing Vpu ORF length does not alter consistent Env expression. (A) Mutations in the env cDNA constructs that removed the upstream Rev and/or Vpu AUG codon (underlined; Rev− and/or Vpu−) or shortened the Vpu ORF at the position corresponding to amino acid 2 (Vpu2) or 35 (ΔU35). Numbers indicate amino acid positions corresponding to the codons. (B and C) HeLa cells were transfected with the env cDNA constructs, pCMVTat2X, pLTR rev1 , and pEGFP. Env and Vpu expression was analyzed by immunoblotting and then densitometry. The percentage of expression relative to that of the env1 R38 mutant (E1R; set at 100%) is graphed for the env isoforms lacking upstream AUG codons (B) or containing altered Vpu ORF lengths (C). wt, wild type; Mk, mock (no env ); E1R to E3R, pDR env1 to pDR env3 cDNA constructs containing the Rev R38 premature termination codon mutation; E2−, env2 cDNA construct expressing the Rev − mutation; E2V−, env2 cDNA construct expressing the Vpu − mutation; E2R−V−, env2 cDNA construct expressing the Rev − and Vpu − mutations; E1RΔU35 to E3RΔU35, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 35; E1RVpu2 to E3RVpu2, env1 to env3 R38 mutant cDNA constructs with the Vpu ORF shortened at the position corresponding to amino acid 2.

    Techniques Used: Expressing, Construct, Transfection, Mutagenesis

    9) 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:

    10) 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

    11) Product Images from "Role of Carbonic Anhydrase IV in Corneal Endothelial HCO3- Transport"

    Article Title: Role of Carbonic Anhydrase IV in Corneal Endothelial HCO3- Transport

    Journal:

    doi: 10.1167/iovs.07-1188

    siRNA knockdown of CAIV. A. Semi-quantitative PCR for CAIV and GAPDH (Glyceraldehyde phosphate dehydrogenase) using untreated, siRNA (20 nM), and siControl (20 nM) treated cultured endothelial cells. B. Western blot for CAIV and βactin using siRNA,
    Figure Legend Snippet: siRNA knockdown of CAIV. A. Semi-quantitative PCR for CAIV and GAPDH (Glyceraldehyde phosphate dehydrogenase) using untreated, siRNA (20 nM), and siControl (20 nM) treated cultured endothelial cells. B. Western blot for CAIV and βactin using siRNA,

    Techniques Used: Real-time Polymerase Chain Reaction, Cell Culture, Western Blot

    A. RT-PCR result for CAIV from bovine lung (positive control), fresh bovine corneal endothelium, and cultured bovine corneal endothelium. B, Western blot of membrane preparations from cultured and fresh bovine corneal endothelium.
    Figure Legend Snippet: A. RT-PCR result for CAIV from bovine lung (positive control), fresh bovine corneal endothelium, and cultured bovine corneal endothelium. B, Western blot of membrane preparations from cultured and fresh bovine corneal endothelium.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Positive Control, Cell Culture, Western Blot

    12) Product Images from "The Role of Prophage for Genome Diversification within a Clonal Lineage of Lactobacillus johnsonii: Characterization of the Defective Prophage LJ771 ▿: Characterization of the Defective Prophage LJ771 ▿ †"

    Article Title: The Role of Prophage for Genome Diversification within a Clonal Lineage of Lactobacillus johnsonii: Characterization of the Defective Prophage LJ771 ▿: Characterization of the Defective Prophage LJ771 ▿ †

    Journal:

    doi: 10.1128/JB.01802-07

    Integration site of prophage LJ771. (A) Sequence alignment of the attL , attR , attB , and attP sites obtained from the sequenced PCR products identified the 33-bp core sequence and a 54-bp repeat region. (B) Deduced N-terminal part of the authentic MsrA
    Figure Legend Snippet: Integration site of prophage LJ771. (A) Sequence alignment of the attL , attR , attB , and attP sites obtained from the sequenced PCR products identified the 33-bp core sequence and a 54-bp repeat region. (B) Deduced N-terminal part of the authentic MsrA

    Techniques Used: Sequencing, Polymerase Chain Reaction

    13) 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

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

    Article Title: Characterization of Glycosomal RING Finger Proteins of Trypanosomatids

    Journal:

    doi: 10.1016/j.exppara.2006.11.004

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

    Techniques Used:

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

    Techniques Used:

    15) Product Images from "Differences in the Mechanism of the Allosteric L-Rhamnose Responses of the AraC/XylS Family Transcription Activators RhaS and RhaR"

    Article Title: Differences in the Mechanism of the Allosteric L-Rhamnose Responses of the AraC/XylS Family Transcription Activators RhaS and RhaR

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2008.06164.x

    Transcription activation by RhaR T279A in RhaR-CTD expressed alone (RhaR(196-311)). Wild-type RhaR(196-311) or RhaR(196-311) T279A (plasmid-encoded) in strain SME3160 (Φ( rhaS - lacZ )Δ85, Δ( rhaSR )::Km) were assayed for transcription
    Figure Legend Snippet: Transcription activation by RhaR T279A in RhaR-CTD expressed alone (RhaR(196-311)). Wild-type RhaR(196-311) or RhaR(196-311) T279A (plasmid-encoded) in strain SME3160 (Φ( rhaS - lacZ )Δ85, Δ( rhaSR )::Km) were assayed for transcription

    Techniques Used: Activation Assay, Plasmid Preparation

    Transcription activation by variants of the RhaS-CTD expressed alone (RhaS(163-278)). Wild-type RhaS(163-278) or variants (plasmid-encoded) in strain SME 3000 (Φ( rhaB-lacZ )Δ84, Δ( rhaSR )::Km) were assayed for transcription activation
    Figure Legend Snippet: Transcription activation by variants of the RhaS-CTD expressed alone (RhaS(163-278)). Wild-type RhaS(163-278) or variants (plasmid-encoded) in strain SME 3000 (Φ( rhaB-lacZ )Δ84, Δ( rhaSR )::Km) were assayed for transcription activation

    Techniques Used: Activation Assay, Plasmid Preparation

    16) Product Images from "Addition of a Single gp120 Glycan Confers Increased Binding to Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin and Neutralization Escape to Human Immunodeficiency Virus Type 1"

    Article Title: Addition of a Single gp120 Glycan Confers Increased Binding to Dendritic Cell-Specific ICAM-3-Grabbing Nonintegrin and Neutralization Escape to Human Immunodeficiency Virus Type 1

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.20.10299-10306.2002

    Increased binding of SHIV SF162P3  envelope gp120 to DC-SIGN-expressing cells. (A) HEK-293T cells transiently expressing human (□) and rhesus (▨) DC-SIGN were incubated with equal amounts (5 ng of p24) of R7/3-33, R7/3-33A, R7/3-162, and R7/3-162P3 viruses, washed, and lysed in 0.5% Triton X-100. The amount of p24 bound was quantified by ELISA and expressed as a percentage of total antigen. Values represent the standard error of the mean from four to five independent experiments. (B) HEK-293T cells were transfected with increasing amounts of the human DC-SIGN plasmid. Transfected cells were incubated with R7/3-162 (□) and R7/3-162P3 (▨) viruses, and the amount of bound p24 was determined. Results are representative of two independent experiments. (C) FACScan analysis of DC-SIGN expression. The mean fluorescence of DC-SIGN monoclonal antibody staining for the transfected cells used in panel B is shown. (D) The binding was performed as described above except that the cells were incubated with mannan (▥, 20 μg/ml) or EGTA (▪, 5 mM) prior to the addition of virus.
    Figure Legend Snippet: Increased binding of SHIV SF162P3 envelope gp120 to DC-SIGN-expressing cells. (A) HEK-293T cells transiently expressing human (□) and rhesus (▨) DC-SIGN were incubated with equal amounts (5 ng of p24) of R7/3-33, R7/3-33A, R7/3-162, and R7/3-162P3 viruses, washed, and lysed in 0.5% Triton X-100. The amount of p24 bound was quantified by ELISA and expressed as a percentage of total antigen. Values represent the standard error of the mean from four to five independent experiments. (B) HEK-293T cells were transfected with increasing amounts of the human DC-SIGN plasmid. Transfected cells were incubated with R7/3-162 (□) and R7/3-162P3 (▨) viruses, and the amount of bound p24 was determined. Results are representative of two independent experiments. (C) FACScan analysis of DC-SIGN expression. The mean fluorescence of DC-SIGN monoclonal antibody staining for the transfected cells used in panel B is shown. (D) The binding was performed as described above except that the cells were incubated with mannan (▥, 20 μg/ml) or EGTA (▪, 5 mM) prior to the addition of virus.

    Techniques Used: Binding Assay, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Fluorescence, Staining

    17) Product Images from "Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae"

    Article Title: Identification of 88 regulatory small RNAs in the TIGR4 strain of the human pathogen Streptococcus pneumoniae

    Journal: RNA

    doi: 10.1261/rna.027359.111

    Detection, genomic context, predicted secondary structure, and target prediction of sRNAs isolated from the S. pneumoniae TIGR4 strain. ( A ) srn491 and ( B ) srn206 . srn206 was detected with a probe labeled internally with biotin, whereas srn491 was hybridized
    Figure Legend Snippet: Detection, genomic context, predicted secondary structure, and target prediction of sRNAs isolated from the S. pneumoniae TIGR4 strain. ( A ) srn491 and ( B ) srn206 . srn206 was detected with a probe labeled internally with biotin, whereas srn491 was hybridized

    Techniques Used: Isolation, Labeling

    srn206 and competence development
    Figure Legend Snippet: srn206 and competence development

    Techniques Used:

    18) Product Images from "HBD-3 structure motifs important in CXCR4 antagonism"

    Article Title: HBD-3 structure motifs important in CXCR4 antagonism

    Journal: The FEBS journal

    doi: 10.1111/febs.12328

    Activity changes resulting from the attachment of α-helix from SDF-1α to the C-terminus of hBD-3
    Figure Legend Snippet: Activity changes resulting from the attachment of α-helix from SDF-1α to the C-terminus of hBD-3

    Techniques Used: Activity Assay

    Determining minimal hBD-3 structure for blocking CXCR4
    Figure Legend Snippet: Determining minimal hBD-3 structure for blocking CXCR4

    Techniques Used: Blocking Assay

    Effects of hBD-3 on saturatable SDF-1α binding to CEM cells
    Figure Legend Snippet: Effects of hBD-3 on saturatable SDF-1α binding to CEM cells

    Techniques Used: Binding Assay

    Effect of Cys mutations on the activity of hBD-3
    Figure Legend Snippet: Effect of Cys mutations on the activity of hBD-3

    Techniques Used: Activity Assay

    Sequence of wild-type (W/T) hBD-3
    Figure Legend Snippet: Sequence of wild-type (W/T) hBD-3

    Techniques Used: Sequencing

    19) Product Images from "The Tropomyosin Binding Region of Cardiac Troponin T Modulates Crossbridge Recruitment Dynamics in Rat Cardiac Muscle Fibers"

    Article Title: The Tropomyosin Binding Region of Cardiac Troponin T Modulates Crossbridge Recruitment Dynamics in Rat Cardiac Muscle Fibers

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2013.01.028

    Normalized pCa-tension relationships in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT
    Figure Legend Snippet: Normalized pCa-tension relationships in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT

    Techniques Used:

    Binding affinity of RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT for Tm
    Figure Legend Snippet: Binding affinity of RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT for Tm

    Techniques Used: Binding Assay

    The rate constant of tension redevelopment, k tr , and the rate constant of lengthmediated XB recruitment, b , in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT
    Figure Legend Snippet: The rate constant of tension redevelopment, k tr , and the rate constant of lengthmediated XB recruitment, b , in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT

    Techniques Used:

    Comparison of far-UV circular dichroism spectral features of RcTnT, RcT1-RfsT2, RfsT1-RcT2, and RfsTnT
    Figure Legend Snippet: Comparison of far-UV circular dichroism spectral features of RcTnT, RcT1-RfsT2, RfsT1-RcT2, and RfsTnT

    Techniques Used:

    SDS-PAGE and Western blot analysis of detergent-skinned papillary muscle fibers reconstituted with RcTnT, RcT1-RfsT2 or RfsT1-RcT2
    Figure Legend Snippet: SDS-PAGE and Western blot analysis of detergent-skinned papillary muscle fibers reconstituted with RcTnT, RcT1-RfsT2 or RfsT1-RcT2

    Techniques Used: SDS Page, Western Blot

    Ca 2+ -activated maximal tension and ATPase activity in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT
    Figure Legend Snippet: Ca 2+ -activated maximal tension and ATPase activity in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT

    Techniques Used: Activity Assay

    Muscle fiber stiffness parameters, E ∞ and E 0 , in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT
    Figure Legend Snippet: Muscle fiber stiffness parameters, E ∞ and E 0 , in rat cardiac muscle fibers reconstituted with RcTnT, RcT1-RfsT2, RfsT1-RcT2 or RfsTnT

    Techniques Used:

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.683201

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

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

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

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

    21) 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

    22) 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

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

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

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00112-10

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

    Techniques Used: Construct

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

    Techniques Used:

    24) Product Images from "Heparin-Binding EGF-Like Growth Factor Is Up-Regulated in the Obstructed Kidney in a Cell- and Region-Specific Manner and Acts to Inhibit Apoptosis"

    Article Title: Heparin-Binding EGF-Like Growth Factor Is Up-Regulated in the Obstructed Kidney in a Cell- and Region-Specific Manner and Acts to Inhibit Apoptosis

    Journal: The American Journal of Pathology

    doi:

    A: Pattern of nucleosomal DNA laddering in transfected and untransfected B7 cells with/without exposure to stretch-stimulation at high frequency and magnitude, and in the presence/absence of CRM197. Nucleosomal DNA laddering was present in untransfected B7 cells following apoptotic stretch-stimulation. In contrast, cells transfected with pro-HB-EGF did not undergo apoptosis in response to apoptotic-stretch. However, in the presence of CRM197 (a specific inhibitor of human HB-EGF), apoptotic stretch was able to induce apoptosis in these cells. B: Quantification of apoptosis using DNA fragmentation enzyme-linked immunosorbent assay in untransfected and transfected B7 cells with/without exposure to stretch-stimulation at high frequency and magnitude, and in the presence/absence of CRM197. Results were obtained from two independent experiments and expressed as a ratio to the values obtained in cells not exposed to stretch.
    Figure Legend Snippet: A: Pattern of nucleosomal DNA laddering in transfected and untransfected B7 cells with/without exposure to stretch-stimulation at high frequency and magnitude, and in the presence/absence of CRM197. Nucleosomal DNA laddering was present in untransfected B7 cells following apoptotic stretch-stimulation. In contrast, cells transfected with pro-HB-EGF did not undergo apoptosis in response to apoptotic-stretch. However, in the presence of CRM197 (a specific inhibitor of human HB-EGF), apoptotic stretch was able to induce apoptosis in these cells. B: Quantification of apoptosis using DNA fragmentation enzyme-linked immunosorbent assay in untransfected and transfected B7 cells with/without exposure to stretch-stimulation at high frequency and magnitude, and in the presence/absence of CRM197. Results were obtained from two independent experiments and expressed as a ratio to the values obtained in cells not exposed to stretch.

    Techniques Used: DNA Laddering, Transfection, Enzyme-linked Immunosorbent Assay

    25) Product Images from "Evolutionarily conserved sequence elements that positively regulate IFN-? expression in T cells"

    Article Title: Evolutionarily conserved sequence elements that positively regulate IFN-? expression in T cells

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

    doi: 10.1073/pnas.0400849101

    Conserved noncoding regions with enhancer activity in the Ifng locus. ( A ) Peaks of similarity in pairwise sequence alignments of the Ifng locus between human and mouse ( Upper ) or human and rat ( Lower ) shown as VISTA plots. Conserved sequences are shown relative to their position in the human genome on the horizontal axes below each panel. The percentage sequence identity is indicated on the vertical axes. Conserved noncoding sequences (CNS) are shown in red, coding exons are shown in blue, and 5′ and 3′ UTRs are shown in turquoise. The position in the mouse genome of the first base of IFNgCNS1, the translation start site, and IFNgCNS2 are shown at the top, where the horizontal arrow indicates the direction of transcription. ( B ) IFNgCNS1 and -2 exhibit enhancer activity. EL-4 cells were transiently transfected with a 9-kb murine Ifng gene alone (mIFNg) or with IFNgCNS1, IFNgCNS2, or both (see Right ) and a β-actin luciferase control plasmid, and either not stimulated (unst) or stimulated with PMA (P), ionomycin (I), or PMA plus ionomycin (P+I), without or with cyclosporin A (CsA). Cells were also cotransfected with a T-bet expression vector or pcDNA. IFN-γ was assessed by ELISA and normalized to luciferase activity [relative light units (RLU)]. The graph is representative of two to five independent experiments with each sample analyzed in duplicate.
    Figure Legend Snippet: Conserved noncoding regions with enhancer activity in the Ifng locus. ( A ) Peaks of similarity in pairwise sequence alignments of the Ifng locus between human and mouse ( Upper ) or human and rat ( Lower ) shown as VISTA plots. Conserved sequences are shown relative to their position in the human genome on the horizontal axes below each panel. The percentage sequence identity is indicated on the vertical axes. Conserved noncoding sequences (CNS) are shown in red, coding exons are shown in blue, and 5′ and 3′ UTRs are shown in turquoise. The position in the mouse genome of the first base of IFNgCNS1, the translation start site, and IFNgCNS2 are shown at the top, where the horizontal arrow indicates the direction of transcription. ( B ) IFNgCNS1 and -2 exhibit enhancer activity. EL-4 cells were transiently transfected with a 9-kb murine Ifng gene alone (mIFNg) or with IFNgCNS1, IFNgCNS2, or both (see Right ) and a β-actin luciferase control plasmid, and either not stimulated (unst) or stimulated with PMA (P), ionomycin (I), or PMA plus ionomycin (P+I), without or with cyclosporin A (CsA). Cells were also cotransfected with a T-bet expression vector or pcDNA. IFN-γ was assessed by ELISA and normalized to luciferase activity [relative light units (RLU)]. The graph is representative of two to five independent experiments with each sample analyzed in duplicate.

    Techniques Used: Activity Assay, Sequencing, Transfection, Luciferase, Plasmid Preparation, Expressing, Enzyme-linked Immunosorbent Assay

    26) Product Images from "Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past"

    Article Title: Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181029

    Zymogram analysis [ 31 , 32 ] of cell extract and supernatant samples of cloned LipJ and Bacillus JR3, performed at 30°C ( A ) and 60°C ( B ). Lane 1: crude cell extract of E . coli expressing LipJ; Lane 2: supernatant of B . cereus 131, used for comparison; Lanes 3–4: soluble (3) and insoluble (4) cell extract of E . coli without the lipJ insert, used as a negative control; Lanes 5–6: concentrated supernatant (5) and crude cell extract (6) of Bacillus strain JR3.
    Figure Legend Snippet: Zymogram analysis [ 31 , 32 ] of cell extract and supernatant samples of cloned LipJ and Bacillus JR3, performed at 30°C ( A ) and 60°C ( B ). Lane 1: crude cell extract of E . coli expressing LipJ; Lane 2: supernatant of B . cereus 131, used for comparison; Lanes 3–4: soluble (3) and insoluble (4) cell extract of E . coli without the lipJ insert, used as a negative control; Lanes 5–6: concentrated supernatant (5) and crude cell extract (6) of Bacillus strain JR3.

    Techniques Used: Clone Assay, Expressing, Negative Control

    Conserved motifs ( A ) and regions ( B ) of short Bacillus and long Geobacillus lipases found in the databases, used for the design of consensus degenerated primers employed for amplification of an internal lipase coding DNA fragment from strain JR3. (C) Tributyrin-supplemented plate assay showing the hydrolysis haloes produced by cloned LipJ (left) and strain JR3 (right).
    Figure Legend Snippet: Conserved motifs ( A ) and regions ( B ) of short Bacillus and long Geobacillus lipases found in the databases, used for the design of consensus degenerated primers employed for amplification of an internal lipase coding DNA fragment from strain JR3. (C) Tributyrin-supplemented plate assay showing the hydrolysis haloes produced by cloned LipJ (left) and strain JR3 (right).

    Techniques Used: Amplification, Produced, Clone Assay

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002042

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

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

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

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

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

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

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

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

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

    Techniques Used: Sequencing

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

    Techniques Used: Sedimentation, Recombinant

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

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

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

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

    28) 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

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

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

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00889-12

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

    Techniques Used: Activation Assay, Binding Assay

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

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

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

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

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

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

    30) Product Images from "The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I 1 Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] [OPEN]"

    Article Title: The Pentatricopeptide Repeat Proteins TANG2 and ORGANELLE TRANSCRIPT PROCESSING439 Are Involved in the Splicing of the Multipartite nad5 Transcript Encoding a Subunit of Mitochondrial Complex I 1 Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] Transcript Encoding a Subunit of Mitochondrial Complex I 1 [W] [OPEN]

    Journal: Plant Physiology

    doi: 10.1104/pp.114.244616

    -PAGE and immunoblot analysis of mitochondrial proteins from tang2 (A and B) and otp439 gels stained for the NADH oxidase activity, and B and D show the polyvinylidene difluoride membranes probed with an anti-Nad9
    Figure Legend Snippet: -PAGE and immunoblot analysis of mitochondrial proteins from tang2 (A and B) and otp439 gels stained for the NADH oxidase activity, and B and D show the polyvinylidene difluoride membranes probed with an anti-Nad9

    Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Activity Assay

    Detailed analysis of nad5 splicing. A, Diagram of the splicing events necessary for generating a translatable nad5 -PCR of the mature and unspliced mRNAs of individual nad5 exons in tang2
    Figure Legend Snippet: Detailed analysis of nad5 splicing. A, Diagram of the splicing events necessary for generating a translatable nad5 -PCR of the mature and unspliced mRNAs of individual nad5 exons in tang2

    Techniques Used: Polymerase Chain Reaction

    -PCR is shown for nad1 , nad2 , nad3 , nad4 , nad5 , nad6 , nad7 , and nad9 transcripts in tang2 , tang2COM plants (A) and in otp439 , otp439COM -PCR of intron-containing mitochondrial
    Figure Legend Snippet: -PCR is shown for nad1 , nad2 , nad3 , nad4 , nad5 , nad6 , nad7 , and nad9 transcripts in tang2 , tang2COM plants (A) and in otp439 , otp439COM -PCR of intron-containing mitochondrial

    Techniques Used: Polymerase Chain Reaction

    insertion, and visible phenotypes of otp439 and tang2 insertion in line SALK_003139 (named tang2 motif of the gene (A), and the insertion in line SALK_089911 (named otp439 ) is in
    Figure Legend Snippet: insertion, and visible phenotypes of otp439 and tang2 insertion in line SALK_003139 (named tang2 motif of the gene (A), and the insertion in line SALK_089911 (named otp439 ) is in

    Techniques Used:

    proteins TANG2 and OTP439 by GFP tagging. GFP fusions were biolistically transformed into Arabidopsis cells (A and D) together with a mitochondria-specific protein (COX4 from S. cerevisiae ) fused to the red fluorescent
    Figure Legend Snippet: proteins TANG2 and OTP439 by GFP tagging. GFP fusions were biolistically transformed into Arabidopsis cells (A and D) together with a mitochondria-specific protein (COX4 from S. cerevisiae ) fused to the red fluorescent

    Techniques Used: Transformation Assay

    31) Product Images from "gon-14 Functions With Class B and Class C Synthetic Multivulva Genes to Control Larval Growth in Caenorhabditis elegans"

    Article Title: gon-14 Functions With Class B and Class C Synthetic Multivulva Genes to Control Larval Growth in Caenorhabditis elegans

    Journal: Genetics

    doi: 10.1534/genetics.105.048751

    GON-14∷VENUS is broadly expressed and predominantly nuclear. (A) Comma stage embryos: GON-14∷VENUS is present in most nuclei. (B) L1 larva: GON-14∷VENUS is present in most nuclei. Inset, nucleus magnified; GON-14∷VENUS localizes to nuclear speckles. (C–E) L4 larva. GON-14 is present in most nuclei, including the somatic gonad (anterior uterus and posterior spermatheca indicated), intestine, and vulva. GON-14∷VENUS is concentrated in nuclear speckles and appears to be largely excluded from nucleoli. (C) Nomarski. (D) Fluorescence. (E) Merge.
    Figure Legend Snippet: GON-14∷VENUS is broadly expressed and predominantly nuclear. (A) Comma stage embryos: GON-14∷VENUS is present in most nuclei. (B) L1 larva: GON-14∷VENUS is present in most nuclei. Inset, nucleus magnified; GON-14∷VENUS localizes to nuclear speckles. (C–E) L4 larva. GON-14 is present in most nuclei, including the somatic gonad (anterior uterus and posterior spermatheca indicated), intestine, and vulva. GON-14∷VENUS is concentrated in nuclear speckles and appears to be largely excluded from nucleoli. (C) Nomarski. (D) Fluorescence. (E) Merge.

    Techniques Used: Fluorescence

    gon-14 larval arrest is suppressed by MES-2,3,6/ESC-E(Z) or MES-4 depletion. (A) gon-14(q12) ; mes-3(RNAi) reaches adulthood when grown at 25°. Arrow, protruding vulva. Bar, 100 μm. (B) gon-14(q12); lin-15B(n744); mes-2(RNAi) reaches adulthood when grown at 20°. Arrow, protruding vulva. Bar, 100 μm. (C) Percentage of adult progeny of the specified genotype. For clarity, gene names are not listed by map position.
    Figure Legend Snippet: gon-14 larval arrest is suppressed by MES-2,3,6/ESC-E(Z) or MES-4 depletion. (A) gon-14(q12) ; mes-3(RNAi) reaches adulthood when grown at 25°. Arrow, protruding vulva. Bar, 100 μm. (B) gon-14(q12); lin-15B(n744); mes-2(RNAi) reaches adulthood when grown at 20°. Arrow, protruding vulva. Bar, 100 μm. (C) Percentage of adult progeny of the specified genotype. For clarity, gene names are not listed by map position.

    Techniques Used:

    gon-14 interacts genetically with the class B and class C synMuv genes to control larval growth. (A) Genes tested for gon-14 interactions, including members of the B and C synMuv classes as well as other genes that interact synthetically with the class B synMuv pathway. Asterisk, predicted protein complexes based on biochemical data from various organisms. See text for references. (B) gon-14(q12) single-mutant adult and gon-14(q12); lin-15B(n744) double-mutant arrested larva, both raised at 20°. Bar, 100 μm. (C) Frequency of larval arrest scored in gene-X single mutants, gon-14 animals, and gene-X; gon-14 animals, where gene-X is a synMuv mutant, a synMuv-interacting mutant, or an RNAi-hypersensitive mutant. gon-14 was disrupted by RNAi or by using a putative null allele, gon-14(q12) , as noted. Double asterisk, progeny scored were descendants from parents heterozygous for the indicated allele. All were raised at 20°.
    Figure Legend Snippet: gon-14 interacts genetically with the class B and class C synMuv genes to control larval growth. (A) Genes tested for gon-14 interactions, including members of the B and C synMuv classes as well as other genes that interact synthetically with the class B synMuv pathway. Asterisk, predicted protein complexes based on biochemical data from various organisms. See text for references. (B) gon-14(q12) single-mutant adult and gon-14(q12); lin-15B(n744) double-mutant arrested larva, both raised at 20°. Bar, 100 μm. (C) Frequency of larval arrest scored in gene-X single mutants, gon-14 animals, and gene-X; gon-14 animals, where gene-X is a synMuv mutant, a synMuv-interacting mutant, or an RNAi-hypersensitive mutant. gon-14 was disrupted by RNAi or by using a putative null allele, gon-14(q12) , as noted. Double asterisk, progeny scored were descendants from parents heterozygous for the indicated allele. All were raised at 20°.

    Techniques Used: Mutagenesis

    gon-14 represses PGL-1 and lag-2∷GFP expression. (A) Wild-type L3 larva, stained with DAPI (white) and antibodies to PGL-1 (green). PGL-1 is restricted to germ cells, outlined by a dashed line. Inset, nucleus is shown; PGL-1 is localized in a punctate perinuclear pattern. (B) Arrested gon-14(q12) larva, stained with DAPI (white) and antibodies to PGL-1 (green). Ectopic PGL-1 is observed in intestinal nuclei (arrowheads). PGL-1 is also observed in germ cells, outlined by a dashed line. Inset, nucleus is shown; somatic PGL-1 is localized in a punctate perinuclear pattern. (C and E) Wild-type L3 grown at 25°. lag-2∷GFP is expressed in the distal tip cell (DTC, arrow) and faintly in the proximal somatic gonad. The gonadal arm is outlined by a dashed line. lag-2∷GFP is not expressed in the intestine (arrowheads). (C) Nomarski. (E) Fluorescence. (D and F) Arrested gon-14(0) larva grown at 25°. lag-2∷GFP is ectopically expressed in the intestine (arrowheads) and faintly expressed in the somatic gonad (arrows). The gonad is outlined by a dashed line. (D) Nomarski. (F) Fluorescence.
    Figure Legend Snippet: gon-14 represses PGL-1 and lag-2∷GFP expression. (A) Wild-type L3 larva, stained with DAPI (white) and antibodies to PGL-1 (green). PGL-1 is restricted to germ cells, outlined by a dashed line. Inset, nucleus is shown; PGL-1 is localized in a punctate perinuclear pattern. (B) Arrested gon-14(q12) larva, stained with DAPI (white) and antibodies to PGL-1 (green). Ectopic PGL-1 is observed in intestinal nuclei (arrowheads). PGL-1 is also observed in germ cells, outlined by a dashed line. Inset, nucleus is shown; somatic PGL-1 is localized in a punctate perinuclear pattern. (C and E) Wild-type L3 grown at 25°. lag-2∷GFP is expressed in the distal tip cell (DTC, arrow) and faintly in the proximal somatic gonad. The gonadal arm is outlined by a dashed line. lag-2∷GFP is not expressed in the intestine (arrowheads). (C) Nomarski. (E) Fluorescence. (D and F) Arrested gon-14(0) larva grown at 25°. lag-2∷GFP is ectopically expressed in the intestine (arrowheads) and faintly expressed in the somatic gonad (arrows). The gonad is outlined by a dashed line. (D) Nomarski. (F) Fluorescence.

    Techniques Used: Expressing, Staining, Fluorescence

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

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

    Journal: Journal of Virology

    doi:

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

    Techniques Used: Immunofluorescence, Infection

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

    Techniques Used: Immunofluorescence, Infection

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

    Techniques Used: Infection

    33) 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

    34) Product Images from "Replacement of the Human Topoisomerase Linker Domain with the Plasmodial Counterpart Renders the Enzyme Camptothecin Resistant"

    Article Title: Replacement of the Human Topoisomerase Linker Domain with the Plasmodial Counterpart Renders the Enzyme Camptothecin Resistant

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068404

    Religation Kinetics. (a) Gel analysis of the religation kinetics observed when incubating the hTop1- or hTop1(pf-Linker)-suicide cleavage complex (Cl1) with the R11 complementary ligator oligonucleotide (shown at the top of the figure) in absence [lanes 3-6 and lanes 12-15 for hTop1 and hTop1(pf-Linker) respectively] or in presence of 100 µM CPT [lanes 7-10 for hTop1 and lanes 16-19 for hTop1(pf-Linker)]. In lane 1 no protein was added. The lanes 2 and 11 represent the time 0 for hTop1 and hTop1(pf-Linker) mediated reactions, respectively. “Cl1” represents the DNA fragment cleaved at the preferred enzyme site; “religation” is the restored fully duplex oligonucleotide representing the final product of the religation reaction. (b) Plot of the percentage of religation normalized to the plateau value, in absence or in presence of CPT for the hTop1 (full and dashed black lines, respectively) and hTop1(pf-Linker) (full and dashed grey lines, respectively). Data shown are means ± SD from 3 independent experiments.
    Figure Legend Snippet: Religation Kinetics. (a) Gel analysis of the religation kinetics observed when incubating the hTop1- or hTop1(pf-Linker)-suicide cleavage complex (Cl1) with the R11 complementary ligator oligonucleotide (shown at the top of the figure) in absence [lanes 3-6 and lanes 12-15 for hTop1 and hTop1(pf-Linker) respectively] or in presence of 100 µM CPT [lanes 7-10 for hTop1 and lanes 16-19 for hTop1(pf-Linker)]. In lane 1 no protein was added. The lanes 2 and 11 represent the time 0 for hTop1 and hTop1(pf-Linker) mediated reactions, respectively. “Cl1” represents the DNA fragment cleaved at the preferred enzyme site; “religation” is the restored fully duplex oligonucleotide representing the final product of the religation reaction. (b) Plot of the percentage of religation normalized to the plateau value, in absence or in presence of CPT for the hTop1 (full and dashed black lines, respectively) and hTop1(pf-Linker) (full and dashed grey lines, respectively). Data shown are means ± SD from 3 independent experiments.

    Techniques Used: Cycling Probe Technology

    Principal Components Analysis. Projection of the motion along the planes formed by the eigenvectors 1 and 3. The data for the hTop1 and hTop1(pf-Linker) proteins are reported in black and red, respectively.
    Figure Legend Snippet: Principal Components Analysis. Projection of the motion along the planes formed by the eigenvectors 1 and 3. The data for the hTop1 and hTop1(pf-Linker) proteins are reported in black and red, respectively.

    Techniques Used:

    Cleavage Kinetics using ribo modified substrate. (a) Sequence of the CL14-U/CP25 substrate and highlight of the Top1 cleavage site containing a scissile ribonucleoside monophosphate. (b) Time course (0.25-15 minutes) of the cleavage reaction of purified hTop1 (lanes 1-8), or hTop1(pf-Linker) (lanes 9-16) with the CL14-U/CP25 substrate (shown in a). In lane 15 the protein has not been added. Cl1 represents the DNA substrate cleaved by the enzymes at the preferred cleavage site. (c) Percentage of cleaved substrate plotted against time for the reaction with hTop1 (black) and with hTop1(pf-Linker) (grey). Data shown are means ± SD from 3 independent experiments.
    Figure Legend Snippet: Cleavage Kinetics using ribo modified substrate. (a) Sequence of the CL14-U/CP25 substrate and highlight of the Top1 cleavage site containing a scissile ribonucleoside monophosphate. (b) Time course (0.25-15 minutes) of the cleavage reaction of purified hTop1 (lanes 1-8), or hTop1(pf-Linker) (lanes 9-16) with the CL14-U/CP25 substrate (shown in a). In lane 15 the protein has not been added. Cl1 represents the DNA substrate cleaved by the enzymes at the preferred cleavage site. (c) Percentage of cleaved substrate plotted against time for the reaction with hTop1 (black) and with hTop1(pf-Linker) (grey). Data shown are means ± SD from 3 independent experiments.

    Techniques Used: Modification, Sequencing, Purification

    Relaxation of supercoiled DNA. Relaxation of negative supercoiled plasmid in a time course experiment for hTop1 and hTop1(pf-Linker) in presence of DMSO (lanes 1–9) and 100µM CPT (lanes 10–18); lane 19, no protein added. The reaction products are resolved in an agarose gel and visualized with ethidium bromide. The two forms of the supercoiled plasmid DNA are indicated as
    Figure Legend Snippet: Relaxation of supercoiled DNA. Relaxation of negative supercoiled plasmid in a time course experiment for hTop1 and hTop1(pf-Linker) in presence of DMSO (lanes 1–9) and 100µM CPT (lanes 10–18); lane 19, no protein added. The reaction products are resolved in an agarose gel and visualized with ethidium bromide. The two forms of the supercoiled plasmid DNA are indicated as "Dimer" and "Monomer".

    Techniques Used: Plasmid Preparation, Cycling Probe Technology, Agarose Gel Electrophoresis

    Root Mean Square Fluctuation. Per-residue Root Mean Square Fluctuations (RMSF) of the hTop1, upper panel black line, and of the hTop1(pf-Linker) model, lower panel red line. In both panels the border of the protein domains is reported.
    Figure Legend Snippet: Root Mean Square Fluctuation. Per-residue Root Mean Square Fluctuations (RMSF) of the hTop1, upper panel black line, and of the hTop1(pf-Linker) model, lower panel red line. In both panels the border of the protein domains is reported.

    Techniques Used:

    Cluster analysis of the linker. Centroids of the clusters representing the families of the linker domain structures for hTop1, left panel, and for the hTop1(pf-Linker), right panel. The structures are colored following the color code blue-white-red, from the most to the less populated cluster.
    Figure Legend Snippet: Cluster analysis of the linker. Centroids of the clusters representing the families of the linker domain structures for hTop1, left panel, and for the hTop1(pf-Linker), right panel. The structures are colored following the color code blue-white-red, from the most to the less populated cluster.

    Techniques Used:

    Cleavage Kinetics using suicide substrate. (a) Time course (0.5-60 minutes) of the cleavage reaction of the purified hTop1 (lanes 1-8), or hTop1(pf-Linker) (lanes 9-16) with the CL14/CP25 suicide substrate, shown at the top of the figure. In lane 17 the protein has not been added. Cl1 represents the DNA substrate cleaved by the enzymes at the preferred cleavage site. (b) Percentage of cleaved suicide substrate plotted against time for the reaction with Top1 (black) and with hTop1(pf-Linker) (grey). Data shown are means ± SD from 3 independent experiments.
    Figure Legend Snippet: Cleavage Kinetics using suicide substrate. (a) Time course (0.5-60 minutes) of the cleavage reaction of the purified hTop1 (lanes 1-8), or hTop1(pf-Linker) (lanes 9-16) with the CL14/CP25 suicide substrate, shown at the top of the figure. In lane 17 the protein has not been added. Cl1 represents the DNA substrate cleaved by the enzymes at the preferred cleavage site. (b) Percentage of cleaved suicide substrate plotted against time for the reaction with Top1 (black) and with hTop1(pf-Linker) (grey). Data shown are means ± SD from 3 independent experiments.

    Techniques Used: Purification

    Cleavage/Religation Equilibrium. (a) Gel electrophoresis of the products generated by incubation of hTop1 (lanes 1-8) and hTop1(pf-Linker) (lanes 9-16) with the [γ - 32 P] end-labelled duplex DNA (CL25/CP25), shown at the top of the figure. The arrow indicates the preferred cleavage site. The duplex was incubated for different time intervals with hTop1 in absence (lanes 1-4) and in presence of CPT (lanes 4-8) or with hTop1(pf-Linker) in absence (lanes 9-12) and in presence of CPT (lanes 13-16). Lane 17, no enzyme added. The band corresponding to the enzyme–substrate cleaved complex, has been indicated by an asterisk. (b) Percentages of the hTop1 (black) or hTop1(pf-Linker) (grey) cleavage complex generated after 1 minute of incubation normalized to the total amount of radiolabeled DNA in each lane. Data shown are means ± SD from 3 independent experiments.
    Figure Legend Snippet: Cleavage/Religation Equilibrium. (a) Gel electrophoresis of the products generated by incubation of hTop1 (lanes 1-8) and hTop1(pf-Linker) (lanes 9-16) with the [γ - 32 P] end-labelled duplex DNA (CL25/CP25), shown at the top of the figure. The arrow indicates the preferred cleavage site. The duplex was incubated for different time intervals with hTop1 in absence (lanes 1-4) and in presence of CPT (lanes 4-8) or with hTop1(pf-Linker) in absence (lanes 9-12) and in presence of CPT (lanes 13-16). Lane 17, no enzyme added. The band corresponding to the enzyme–substrate cleaved complex, has been indicated by an asterisk. (b) Percentages of the hTop1 (black) or hTop1(pf-Linker) (grey) cleavage complex generated after 1 minute of incubation normalized to the total amount of radiolabeled DNA in each lane. Data shown are means ± SD from 3 independent experiments.

    Techniques Used: Nucleic Acid Electrophoresis, Generated, Incubation, Cycling Probe Technology

    35) Product Images from "RTX Toxin Plays a Key Role in Kingella kingae Virulence in an Infant Rat Model"

    Article Title: RTX Toxin Plays a Key Role in Kingella kingae Virulence in an Infant Rat Model

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01636-14

    In vitro assay of K. kingae toxicity. For toxicity tests, 1.0 × 10 5 PYKK081 cells were added to 0.5 × 10 6 THP-1 cells in RPMI with 10% FBS. Each time point was assayed in duplicate in three independent experiments, and samples were collected every 30 min for 3 h. The data in the figure are representative of the three experiments. The cell viability was estimated by ATP production, which was detected using the CellTiter-Glo luminescence assay. Relative viability refers to the PYKK081 luminescence value divided by the KKNB100 luminescence value at a given time point. THP-1 negative-control wells at each time point were consistently at or slightly below the luminescence of KKNB100. For the toxin accumulation experiment, the protein was precipitated from 200 μl of THP-1 culture medium before loading on the gel. The presence of toxin from PYKK081 was identified by Western blotting using 10A7D7 antibody. The Western blot shown contains the samples taken from the relative viability experiment described above. The correlation is representative of those in the other experiments.
    Figure Legend Snippet: In vitro assay of K. kingae toxicity. For toxicity tests, 1.0 × 10 5 PYKK081 cells were added to 0.5 × 10 6 THP-1 cells in RPMI with 10% FBS. Each time point was assayed in duplicate in three independent experiments, and samples were collected every 30 min for 3 h. The data in the figure are representative of the three experiments. The cell viability was estimated by ATP production, which was detected using the CellTiter-Glo luminescence assay. Relative viability refers to the PYKK081 luminescence value divided by the KKNB100 luminescence value at a given time point. THP-1 negative-control wells at each time point were consistently at or slightly below the luminescence of KKNB100. For the toxin accumulation experiment, the protein was precipitated from 200 μl of THP-1 culture medium before loading on the gel. The presence of toxin from PYKK081 was identified by Western blotting using 10A7D7 antibody. The Western blot shown contains the samples taken from the relative viability experiment described above. The correlation is representative of those in the other experiments.

    Techniques Used: In Vitro, Luminescence Assay, Negative Control, Western Blot

    Kaplan-Meier survival curves of rat offspring after i.p. injections with K. kingae . PN 21 or PN 7 rats were injected with different doses of K. kingae strains or PBS. The animals were monitored for mortality and clinical signs of illness twice a day for 14 days. After the initial 72 h, no additional mortality was detected. These data were combined from three separate experiments. (A) The survival curve of 21-day-old rats injected with 1.3 × 10 8 bacterial cells/animal of either PYKK081 or KKNB100 demonstrated no significant difference in mortality (log rank test, P > 0.05). (B) The survival curves of 7-day-old rats injected with 4.5 × 10 7 , 2.0 × 10 7 , and 1.2 × 10 7 bacterial cells/animal all demonstrated significant difference in mortality when comparing PYKK081 and KKNB100. Additionally, survival curves were significantly dependent upon the concentration of injected PYKK081 (log rank test for trend, P
    Figure Legend Snippet: Kaplan-Meier survival curves of rat offspring after i.p. injections with K. kingae . PN 21 or PN 7 rats were injected with different doses of K. kingae strains or PBS. The animals were monitored for mortality and clinical signs of illness twice a day for 14 days. After the initial 72 h, no additional mortality was detected. These data were combined from three separate experiments. (A) The survival curve of 21-day-old rats injected with 1.3 × 10 8 bacterial cells/animal of either PYKK081 or KKNB100 demonstrated no significant difference in mortality (log rank test, P > 0.05). (B) The survival curves of 7-day-old rats injected with 4.5 × 10 7 , 2.0 × 10 7 , and 1.2 × 10 7 bacterial cells/animal all demonstrated significant difference in mortality when comparing PYKK081 and KKNB100. Additionally, survival curves were significantly dependent upon the concentration of injected PYKK081 (log rank test for trend, P

    Techniques Used: Injection, Concentration Assay

    Gross pathology and histopathological examination of infant rats injected with K. kingae . The animals were i.p. injected with 2.0 × 10 7 cells of PYKK081 or KKNB100 or with PBS and sacrificed 48 h after injection for tissue analysis. Representative pictures are shown. (A) Ventral view of the animals injected with PYKK081 (left), exhibiting an abdominal necrotic lesion and significant weight loss, KKNB100 (middle), and PBS (right). (B) Magnified view of the skin lesion formed as a result of i.p. injection of PYKK081, which demonstrated considerable necrosis, and associated acute inflammation involving the panniculus carnosus muscularis (PCM), the subdermal tissues, and presumably adherent portions of peritoneum. (C) Skin lesion section from the animal injected with PYKK081, which demonstrates muscle necrosis, some associated acute inflammation, and bacteria. Magnification, ×1,000. (D) Slide from the animal injected with KKNB100, which shows no bacteria or any histopathological changes. Magnification, ×400. (E) Skin lesion section seen in panel C. Magnification, ×400. (F to I) Immunohistochemical analysis of the skin lesion slides was performed using anti- K. kingae OMV antibody and was visualized upon interaction with Alexa 488-conjugated anti-rabbit IgG. Magnification, ×400. Nonspecific fluorescence was not observed in the absence of primary antibody. Cell nuclei were labeled with 7-AAD. An intense green signal, indicating the presence of K. kingae , was clearly observed between the abdominal thin muscle layer underlying the skin and the fatty layer in the samples from animals injected with PYKK081. (F) Skin lesion section from the animal injected with PYKK081. A mixture of intracellular organisms and extensive extracellular K. kingae was detected. (G) Lesion section from the animal injected with PYKK081. The bacteria are predominantly found inside phagocytic cells. (H) In the skin lesion section from the animal injected with KKNB100, the majority of staining was observed in phagocytic cells. The samples show much less staining. (I) Site of injection from the animal injected with PBS. No green signal was detected in these samples.
    Figure Legend Snippet: Gross pathology and histopathological examination of infant rats injected with K. kingae . The animals were i.p. injected with 2.0 × 10 7 cells of PYKK081 or KKNB100 or with PBS and sacrificed 48 h after injection for tissue analysis. Representative pictures are shown. (A) Ventral view of the animals injected with PYKK081 (left), exhibiting an abdominal necrotic lesion and significant weight loss, KKNB100 (middle), and PBS (right). (B) Magnified view of the skin lesion formed as a result of i.p. injection of PYKK081, which demonstrated considerable necrosis, and associated acute inflammation involving the panniculus carnosus muscularis (PCM), the subdermal tissues, and presumably adherent portions of peritoneum. (C) Skin lesion section from the animal injected with PYKK081, which demonstrates muscle necrosis, some associated acute inflammation, and bacteria. Magnification, ×1,000. (D) Slide from the animal injected with KKNB100, which shows no bacteria or any histopathological changes. Magnification, ×400. (E) Skin lesion section seen in panel C. Magnification, ×400. (F to I) Immunohistochemical analysis of the skin lesion slides was performed using anti- K. kingae OMV antibody and was visualized upon interaction with Alexa 488-conjugated anti-rabbit IgG. Magnification, ×400. Nonspecific fluorescence was not observed in the absence of primary antibody. Cell nuclei were labeled with 7-AAD. An intense green signal, indicating the presence of K. kingae , was clearly observed between the abdominal thin muscle layer underlying the skin and the fatty layer in the samples from animals injected with PYKK081. (F) Skin lesion section from the animal injected with PYKK081. A mixture of intracellular organisms and extensive extracellular K. kingae was detected. (G) Lesion section from the animal injected with PYKK081. The bacteria are predominantly found inside phagocytic cells. (H) In the skin lesion section from the animal injected with KKNB100, the majority of staining was observed in phagocytic cells. The samples show much less staining. (I) Site of injection from the animal injected with PBS. No green signal was detected in these samples.

    Techniques Used: Injection, Immunohistochemistry, Fluorescence, Labeling, Staining

    Mutagenesis of rtxA and genetic complementation. (A) The schematic presentation of the rtx operon demonstrates the aphA3 kanamycin cassette insertion in the rtxA gene of KKNB100. The positions of the primers used for qPCR analysis of K. kingae are shown. (B) Schematic presentation of the genetic construct used for chromosome-based genetic complementation of the rtxA gene of KKNB100. (C) Purification of RtxA from the PYKK081 secreted fraction. The PYKK081 secreted fraction was isolated and subjected to G-100 chromatography as described in Materials and Methods. The samples were resolved by SDS-PAGE, and the proteins were stained with Coomassie blue. The 100-kDa protein band was eluted from the gel by electroelution. The purified RtxA was used as an antigen to obtain anti-RtxA monoclonal antibody. The protein ladder is shown in lane L, G-100-purified proteins (125 μg of protein loaded) are shown in lane 1, and the RtxA band (10 μg of protein loaded) after electroelution is shown in lane 2. (D) Determination of cytoxicity for PYKK081 and genetic constructs. For toxicity tests, 1 × 10 5 K. kingae cells were added to 0.5 × 10 6 THP-1 cells in RPMI with 10% FBS. Cell viability was estimated by ATP production, which was detected using the CellTiter-Glo luminescence assay. Each condition was assayed in duplicate, and luminescence values were recorded after 3 h. Mean values from three independent experiments are displayed. A one-way ANOVA was preformed among the three conditions; relative cell viability differed significantly ( P
    Figure Legend Snippet: Mutagenesis of rtxA and genetic complementation. (A) The schematic presentation of the rtx operon demonstrates the aphA3 kanamycin cassette insertion in the rtxA gene of KKNB100. The positions of the primers used for qPCR analysis of K. kingae are shown. (B) Schematic presentation of the genetic construct used for chromosome-based genetic complementation of the rtxA gene of KKNB100. (C) Purification of RtxA from the PYKK081 secreted fraction. The PYKK081 secreted fraction was isolated and subjected to G-100 chromatography as described in Materials and Methods. The samples were resolved by SDS-PAGE, and the proteins were stained with Coomassie blue. The 100-kDa protein band was eluted from the gel by electroelution. The purified RtxA was used as an antigen to obtain anti-RtxA monoclonal antibody. The protein ladder is shown in lane L, G-100-purified proteins (125 μg of protein loaded) are shown in lane 1, and the RtxA band (10 μg of protein loaded) after electroelution is shown in lane 2. (D) Determination of cytoxicity for PYKK081 and genetic constructs. For toxicity tests, 1 × 10 5 K. kingae cells were added to 0.5 × 10 6 THP-1 cells in RPMI with 10% FBS. Cell viability was estimated by ATP production, which was detected using the CellTiter-Glo luminescence assay. Each condition was assayed in duplicate, and luminescence values were recorded after 3 h. Mean values from three independent experiments are displayed. A one-way ANOVA was preformed among the three conditions; relative cell viability differed significantly ( P

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, Construct, Purification, Isolation, Chromatography, SDS Page, Staining, Luminescence Assay

    Histopathological examination rat organs. Hematoxylin-eosin stain preparation of organ sections (original magnification, ×100) obtained from autopsied PN 9 rats at 48 h postinjection with PYKK081 (A) or KKNB100/PBS (B). At least four animals from each strain group were sampled for histological analysis. Representative slides are shown.
    Figure Legend Snippet: Histopathological examination rat organs. Hematoxylin-eosin stain preparation of organ sections (original magnification, ×100) obtained from autopsied PN 9 rats at 48 h postinjection with PYKK081 (A) or KKNB100/PBS (B). At least four animals from each strain group were sampled for histological analysis. Representative slides are shown.

    Techniques Used: Staining

    36) Product Images from "Horizontal gene transfer from flowering plants to Gnetum"

    Article Title: Horizontal gene transfer from flowering plants to Gnetum

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

    doi: 10.1073/pnas.1833775100

    Relative location of the five nad1 exons in the Petunia mt genome and a predicted secondary structure of the gymnosperm-type mt nad1 intron 2 of Gnetum gnemonoides .( A ) Arrows indicate the 5′ → 3′ orientation of the nad1 exon regions. An asterisk in the intron 2 scheme indicates the intervening nucleotides of the Gnetum gymnosperm-type intron. Mitochondrial map modified from Conklin et al. ). ( B ) Domain lengths before the slash refer to Gnetum gymnosperm-type introns, those after the slash to Gnetum angiosperm-type introns.
    Figure Legend Snippet: Relative location of the five nad1 exons in the Petunia mt genome and a predicted secondary structure of the gymnosperm-type mt nad1 intron 2 of Gnetum gnemonoides .( A ) Arrows indicate the 5′ → 3′ orientation of the nad1 exon regions. An asterisk in the intron 2 scheme indicates the intervening nucleotides of the Gnetum gymnosperm-type intron. Mitochondrial map modified from Conklin et al. ). ( B ) Domain lengths before the slash refer to Gnetum gymnosperm-type introns, those after the slash to Gnetum angiosperm-type introns.

    Techniques Used: Modification

    Distribution of mt nad1 partial exons b and c and intron 2, and age estimates for Gnetum crown groups. ( A ) Bootstrap majority-rule consensus tree obtained from conserved intron and exon sections analyzed by neighbor-joining, using GTR + G + I distances (α = 0.097 and P inv = 0.120). ( B ) Distribution of the gymnosperm- and angiosperm-type introns on the maximum likelihood tree obtained from concatenated Gnetum nuclear and plastid sequences. Filled squares indicate intron presence; hatched squares represent partially sequenced or PCR-verified accessions; open squares indicate lack of intron. No introns were detected in G. africanum and Welwitschia . Numbers next to nodes represent parsimony bootstrap support, followed by Bayesian posterior probabilities. ( C ) Age estimates for Gnetum crown groups from chloroplast matK and rbcL sequences. Arrows indicate nodes constrained by fossils. a, age of Gnetales (205–125 my, depending on the fossil used); b, age of the Gnetum - Welwitschia divergence (115–70 my); c, age of extant Gnetum (11–6 my); d, age of Asian Gnetum ; e, age of Asian clade II Gnetum .
    Figure Legend Snippet: Distribution of mt nad1 partial exons b and c and intron 2, and age estimates for Gnetum crown groups. ( A ) Bootstrap majority-rule consensus tree obtained from conserved intron and exon sections analyzed by neighbor-joining, using GTR + G + I distances (α = 0.097 and P inv = 0.120). ( B ) Distribution of the gymnosperm- and angiosperm-type introns on the maximum likelihood tree obtained from concatenated Gnetum nuclear and plastid sequences. Filled squares indicate intron presence; hatched squares represent partially sequenced or PCR-verified accessions; open squares indicate lack of intron. No introns were detected in G. africanum and Welwitschia . Numbers next to nodes represent parsimony bootstrap support, followed by Bayesian posterior probabilities. ( C ) Age estimates for Gnetum crown groups from chloroplast matK and rbcL sequences. Arrows indicate nodes constrained by fossils. a, age of Gnetales (205–125 my, depending on the fossil used); b, age of the Gnetum - Welwitschia divergence (115–70 my); c, age of extant Gnetum (11–6 my); d, age of Asian Gnetum ; e, age of Asian clade II Gnetum .

    Techniques Used: Polymerase Chain Reaction

    37) Product Images from "bHLH003, bHLH013 and bHLH017 Are New Targets of JAZ Repressors Negatively Regulating JA Responses"

    Article Title: bHLH003, bHLH013 and bHLH017 Are New Targets of JAZ Repressors Negatively Regulating JA Responses

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086182

    bHLH003, bHLH013 and bHLH017 interact with JAZ and can form homo and heterodimers. (A) Yeast cells co-transformed with the indicated combinations of JAZ-BD (pGBKT7) and bHLH-AD (pGADT7), grown for 3 days in medium lacking Ade, His, Leu and Trp to select for interactions. Numbers represent the number of JAZ protein (JAZ1 to JAZ12) and C represents the empty pGBKT7 vector containing only the BD. Transformation controls are shown in Figure S1 . (B) Immunoblots with anti-HA antibody of the bHLH003-HA, bHLH013-HA and bHLH017-HA from transgenic plant extracts, pulled down by recombinant MBP-JAZ proteins expressed in E.coli . WT extracts were used as control (c). The two first lanes of each blot correspond to 40 µl of the input protein extract used for pull-down. Bellow each immunoblot, a coomassie stained gel shows the amount of recombinant MBP-fused protein used in each sample. (C) Immunoblots of pulled-down bHLH-HA proteins from transgenic plant extracts, by recombinant MBP-bHLH, presented as in (B) except that 30 µl from the total plant extract were loaded in the first lane of each gel. MBP controls are common for assays in (B) and (C) since both were done simultaneously.
    Figure Legend Snippet: bHLH003, bHLH013 and bHLH017 interact with JAZ and can form homo and heterodimers. (A) Yeast cells co-transformed with the indicated combinations of JAZ-BD (pGBKT7) and bHLH-AD (pGADT7), grown for 3 days in medium lacking Ade, His, Leu and Trp to select for interactions. Numbers represent the number of JAZ protein (JAZ1 to JAZ12) and C represents the empty pGBKT7 vector containing only the BD. Transformation controls are shown in Figure S1 . (B) Immunoblots with anti-HA antibody of the bHLH003-HA, bHLH013-HA and bHLH017-HA from transgenic plant extracts, pulled down by recombinant MBP-JAZ proteins expressed in E.coli . WT extracts were used as control (c). The two first lanes of each blot correspond to 40 µl of the input protein extract used for pull-down. Bellow each immunoblot, a coomassie stained gel shows the amount of recombinant MBP-fused protein used in each sample. (C) Immunoblots of pulled-down bHLH-HA proteins from transgenic plant extracts, by recombinant MBP-bHLH, presented as in (B) except that 30 µl from the total plant extract were loaded in the first lane of each gel. MBP controls are common for assays in (B) and (C) since both were done simultaneously.

    Techniques Used: Transformation Assay, Plasmid Preparation, Western Blot, Transgenic Assay, Recombinant, Staining

    Sub-cellular localization of bHLH003, bHLH013 and bHLH017. Microscopy images of GFP signal detected in root cells of transgenic Arabidopsis transgenic plants over-expressing bHLH003-GFP, bHLH013-GFP or bHLH017-GFP and grown for ten days in media supplemented (A) or not (B) with 50 µM JA for 3 h.
    Figure Legend Snippet: Sub-cellular localization of bHLH003, bHLH013 and bHLH017. Microscopy images of GFP signal detected in root cells of transgenic Arabidopsis transgenic plants over-expressing bHLH003-GFP, bHLH013-GFP or bHLH017-GFP and grown for ten days in media supplemented (A) or not (B) with 50 µM JA for 3 h.

    Techniques Used: Microscopy, Transgenic Assay, Expressing

    DNA-binding specificity of bHLH003, bHLH013 and bHLH017. (A) Position weight matrix (PWM) representation of the three top scoring 8-mers obtained in “seed-and-wobble” algorithm corresponding to bHLH003, bHLH013 and bHLH017. All three proteins showed highest binding affinity to a canonical G-box (CACGTG). (B) Median enrichment scores (E-scores) of the all the possible G-containing 8mers flanked by purine (R) and/or pyrimidine (Y) nucleotides recognized by the three bHLH proteins tested. bHLH003 showed a strong dependence for purine at 5′-end and pyrimidine at 3′-end. We included for comparison data corresponding to MYC2 previously described [53] . (C) Box-plot of E-scores of G-related variants. Boxes represent quartiles 25% to 75%, and black line represents the median of the distribution (quartile 50%). Bars indicate quartiles 1 to 25% (above) and 75 to 100% (below), and dots denote outliers of the distribution. Data corresponding to MYC2 were previously described [53] .
    Figure Legend Snippet: DNA-binding specificity of bHLH003, bHLH013 and bHLH017. (A) Position weight matrix (PWM) representation of the three top scoring 8-mers obtained in “seed-and-wobble” algorithm corresponding to bHLH003, bHLH013 and bHLH017. All three proteins showed highest binding affinity to a canonical G-box (CACGTG). (B) Median enrichment scores (E-scores) of the all the possible G-containing 8mers flanked by purine (R) and/or pyrimidine (Y) nucleotides recognized by the three bHLH proteins tested. bHLH003 showed a strong dependence for purine at 5′-end and pyrimidine at 3′-end. We included for comparison data corresponding to MYC2 previously described [53] . (C) Box-plot of E-scores of G-related variants. Boxes represent quartiles 25% to 75%, and black line represents the median of the distribution (quartile 50%). Bars indicate quartiles 1 to 25% (above) and 75 to 100% (below), and dots denote outliers of the distribution. Data corresponding to MYC2 were previously described [53] .

    Techniques Used: Binding Assay

    bHLH003, bHLH013 and bHLH017 are transcriptional repressors. (A) Schematic representation of reporter and effector constructs used in transient expression experiments in Nicotiana benthamiana . The reporter is the fusion of the JAZ2 promoter to firefly LUC coding sequence. MYC2, bHLH003, bHLH013 and bHLH017 genes expressed under the CaMV 35S promoter were used as effectors. (B) Induction or repression of pJAZ2:LUC reporter by MYC2, bHLH003, bHLH013 and bHLH017. Error bars indicate the SE from 16 replicates. (C) Effect of bHLH003 or bHLH017 expression on the MYC2 transactivation activity of pJAZ2:LUC . Error bars indicate the SE of results of 16 replicates. Asterisks represent p
    Figure Legend Snippet: bHLH003, bHLH013 and bHLH017 are transcriptional repressors. (A) Schematic representation of reporter and effector constructs used in transient expression experiments in Nicotiana benthamiana . The reporter is the fusion of the JAZ2 promoter to firefly LUC coding sequence. MYC2, bHLH003, bHLH013 and bHLH017 genes expressed under the CaMV 35S promoter were used as effectors. (B) Induction or repression of pJAZ2:LUC reporter by MYC2, bHLH003, bHLH013 and bHLH017. Error bars indicate the SE from 16 replicates. (C) Effect of bHLH003 or bHLH017 expression on the MYC2 transactivation activity of pJAZ2:LUC . Error bars indicate the SE of results of 16 replicates. Asterisks represent p

    Techniques Used: Construct, Expressing, Sequencing, Activity Assay

    Responses to JA are affected in bHLH mutant and over-expression (OE) lines. (A) Root growth inhibition by JA (10 µM) of 8 days-old wild-type (WT), bhlh003 and bhlh017 mutant seedlings and OE lines of bHLH003, bHLH013 and bHLH017 grown in vertical MS plates. Values correspond to the average root growth betwen day 2 and day 8. Error bars represent standard deviations. Asterisks represents values that are significantly different statistically from WT applying a Student's t-test (p
    Figure Legend Snippet: Responses to JA are affected in bHLH mutant and over-expression (OE) lines. (A) Root growth inhibition by JA (10 µM) of 8 days-old wild-type (WT), bhlh003 and bhlh017 mutant seedlings and OE lines of bHLH003, bHLH013 and bHLH017 grown in vertical MS plates. Values correspond to the average root growth betwen day 2 and day 8. Error bars represent standard deviations. Asterisks represents values that are significantly different statistically from WT applying a Student's t-test (p

    Techniques Used: Mutagenesis, Over Expression, Inhibition, Mass Spectrometry

    Schematic model of the role of bHLHs within the JA signaling pathway. In the absence of JA-Ile, JAZ repressors are stable and interact with MYC2, MYC3 and MYC4 as well as bHLH003, bHLH013 and bHLH017. JAZs are part of a repression complex that comprises NINJA and TOPLESS (not shown). Upon a stimulus, hormone is perceived by the COI1/JAZ co-receptor and JAZ proteins are targeted for degradation by the proteasome. Once released from JAZ, MYC2, MYC3 and MYC4 will activate transcription, whereas bHLH003, bHLH013 and bHLH017 will repress it. Both sets of proteins (MYCs and bHLHs) compete for the G-box and, therefore, the output response will depend on the balance of activity between these two sets of TFs. Moreover, bHLH017 expression is activated by MYC2, therefore increasing the effective repressor over time and further contributing to reduce transcriptional activation.
    Figure Legend Snippet: Schematic model of the role of bHLHs within the JA signaling pathway. In the absence of JA-Ile, JAZ repressors are stable and interact with MYC2, MYC3 and MYC4 as well as bHLH003, bHLH013 and bHLH017. JAZs are part of a repression complex that comprises NINJA and TOPLESS (not shown). Upon a stimulus, hormone is perceived by the COI1/JAZ co-receptor and JAZ proteins are targeted for degradation by the proteasome. Once released from JAZ, MYC2, MYC3 and MYC4 will activate transcription, whereas bHLH003, bHLH013 and bHLH017 will repress it. Both sets of proteins (MYCs and bHLHs) compete for the G-box and, therefore, the output response will depend on the balance of activity between these two sets of TFs. Moreover, bHLH017 expression is activated by MYC2, therefore increasing the effective repressor over time and further contributing to reduce transcriptional activation.

    Techniques Used: Activity Assay, Expressing, Activation Assay

    Expression patterns of bHLH003, bHLH013 and bHLH017 in Arabidopsis. Histochemical GUS staining of 6 days-old or adult Arabidopsis plants expressing the GUS reporter under the control of the bHLH003, bHLH013 and bHLH017 promoters. From upper to bottom panels, GUS activity was detected in cotyledon, roots, adult leaves, anthers, sepals, petals and pistil. GUS activity was detected in cotyledon and leaf, roots, petals, sepals, etc, etc.
    Figure Legend Snippet: Expression patterns of bHLH003, bHLH013 and bHLH017 in Arabidopsis. Histochemical GUS staining of 6 days-old or adult Arabidopsis plants expressing the GUS reporter under the control of the bHLH003, bHLH013 and bHLH017 promoters. From upper to bottom panels, GUS activity was detected in cotyledon, roots, adult leaves, anthers, sepals, petals and pistil. GUS activity was detected in cotyledon and leaf, roots, petals, sepals, etc, etc.

    Techniques Used: Expressing, Staining, Activity Assay

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

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

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erq234

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

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

    39) Product Images from "A Cyclin-Dependent Kinase that Promotes Cytokinesis through Modulating Phosphorylation of the Carboxy Terminal Domain of the RNA Pol II Rpb1p Sub-Unit"

    Article Title: A Cyclin-Dependent Kinase that Promotes Cytokinesis through Modulating Phosphorylation of the Carboxy Terminal Domain of the RNA Pol II Rpb1p Sub-Unit

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000433

    Lsk1p and Lsc1p co-localize to the nucleus and physically interact in a manner dependent on Lsk1p kinase activity. (A) Cells expressing Lsk1-CFP or Lsc1-GFP under the control of the thiamine repressible nmt1 promoter were grown for 12 hours in minimal media in the absence of thiamine, and then imaged in the CFP and YFP channels, respectively. Bar, 3 µM. (B) Wild-type, Lsk1-HA, Lsc1-myc, and Lsk1-HA Lsc1-myc cells were grown to mid-log phase and lysed under native conditions. A portion of the lysates were subjected to anti-HA and anti-myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the myc epitope. (C) Cells of the indicated genotype, carrying integrated GFP tagged versions of Lsk1p or Lsc1p under the control of their native promoter, were grown to mid-log phase in YES media, fixed, and then stained with DAPI (nuclei) and antibodies specific for microtubules and GFP. Bar, 3 µM. (D) Lsk1-HA, Lsk1-K306R-HA, Lsk1-HA Lsc1-myc, and Lsk1-K306R-HA Lsc1-myc cells were grown to mid-log phase in YES and lysed under native conditions. A portion of the lysates were subjected to anti-HA and anti-myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the myc epitope.
    Figure Legend Snippet: Lsk1p and Lsc1p co-localize to the nucleus and physically interact in a manner dependent on Lsk1p kinase activity. (A) Cells expressing Lsk1-CFP or Lsc1-GFP under the control of the thiamine repressible nmt1 promoter were grown for 12 hours in minimal media in the absence of thiamine, and then imaged in the CFP and YFP channels, respectively. Bar, 3 µM. (B) Wild-type, Lsk1-HA, Lsc1-myc, and Lsk1-HA Lsc1-myc cells were grown to mid-log phase and lysed under native conditions. A portion of the lysates were subjected to anti-HA and anti-myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the myc epitope. (C) Cells of the indicated genotype, carrying integrated GFP tagged versions of Lsk1p or Lsc1p under the control of their native promoter, were grown to mid-log phase in YES media, fixed, and then stained with DAPI (nuclei) and antibodies specific for microtubules and GFP. Bar, 3 µM. (D) Lsk1-HA, Lsk1-K306R-HA, Lsk1-HA Lsc1-myc, and Lsk1-K306R-HA Lsc1-myc cells were grown to mid-log phase in YES and lysed under native conditions. A portion of the lysates were subjected to anti-HA and anti-myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the myc epitope.

    Techniques Used: Activity Assay, Expressing, SDS Page, Staining

    Strains bearing the lsc1Δ or lsk1-K306R mutations display highly similar cytokinesis defects as compared to lsk1Δ mutants. (A) A phylogeny analyzing the relationship between S. pombe cyclins (black type) and the cyclin partners of Lsk1p relatives in budding yeast, mouse, human, and Drosophila (blue type). The most likely cyclin partner of Lsk1p based on this analysis, SPCC530.13, is shown in red. The phylogeny was created by MEGA version 3.1 software using the neighbor-joining algorithm. Sp, Schizosacharomyces pombe ; Dm, Drosophila melanogaster ; Hs Homo sapiens ; Mm Mus musculus ; Sc Saccharomyces cerevisiae . (B) ClustalW alignment comparing S. pombe SPCC530.13, S. cerevisiae Ctk2p, and human cyclin T. (C) Ten-fold serial dilutions of logarithmically growing cultures were plated onto YES plates containing 0.5 µM LatA or DMSO (solvent control) and incubated at 32°C for 3 days. (D) Cells of the indicated genotype were grown to mid-log phase at 32°C and then treated with 0.3 µM LatA for 5 hours before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µM. (E) Cells of the indicated genotype were freshly streaked to YES plates and incubated for 24 hours at 36°C. Bar, 50 µM.
    Figure Legend Snippet: Strains bearing the lsc1Δ or lsk1-K306R mutations display highly similar cytokinesis defects as compared to lsk1Δ mutants. (A) A phylogeny analyzing the relationship between S. pombe cyclins (black type) and the cyclin partners of Lsk1p relatives in budding yeast, mouse, human, and Drosophila (blue type). The most likely cyclin partner of Lsk1p based on this analysis, SPCC530.13, is shown in red. The phylogeny was created by MEGA version 3.1 software using the neighbor-joining algorithm. Sp, Schizosacharomyces pombe ; Dm, Drosophila melanogaster ; Hs Homo sapiens ; Mm Mus musculus ; Sc Saccharomyces cerevisiae . (B) ClustalW alignment comparing S. pombe SPCC530.13, S. cerevisiae Ctk2p, and human cyclin T. (C) Ten-fold serial dilutions of logarithmically growing cultures were plated onto YES plates containing 0.5 µM LatA or DMSO (solvent control) and incubated at 32°C for 3 days. (D) Cells of the indicated genotype were grown to mid-log phase at 32°C and then treated with 0.3 µM LatA for 5 hours before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µM. (E) Cells of the indicated genotype were freshly streaked to YES plates and incubated for 24 hours at 36°C. Bar, 50 µM.

    Techniques Used: Software, Incubation, Staining

    40) Product Images from "Identification of a Novel Enzyme Required for Starch Metabolism in Arabidopsis Leaves. The Phosphoglucan, Water Dikinase 1Identification of a Novel Enzyme Required for Starch Metabolism in Arabidopsis Leaves. The Phosphoglucan, Water Dikinase 1 [w]"

    Article Title: Identification of a Novel Enzyme Required for Starch Metabolism in Arabidopsis Leaves. The Phosphoglucan, Water Dikinase 1Identification of a Novel Enzyme Required for Starch Metabolism in Arabidopsis Leaves. The Phosphoglucan, Water Dikinase 1 [w]

    Journal: Plant Physiology

    doi: 10.1104/pp.104.055954

    OK1 Is a Phosphoglucan, Water Dikinase
    Figure Legend Snippet: OK1 Is a Phosphoglucan, Water Dikinase

    Techniques Used:

    OK1 contains a starch binding domain and domains typical for dikinases. A, Protein domains of the OK1 protein. The amino acid number indicates the location of the respective domain within the protein. CTS, Chloroplast targeting signal; SB, starch binding
    Figure Legend Snippet: OK1 contains a starch binding domain and domains typical for dikinases. A, Protein domains of the OK1 protein. The amino acid number indicates the location of the respective domain within the protein. CTS, Chloroplast targeting signal; SB, starch binding

    Techniques Used: Binding Assay

    OK1 is located in chloroplasts. Equal amounts of protein extracted from Arabidopsis leaf protoplasts (P) and chloroplasts (C), respectively, were separated by SDS-PAGE and examined by immunoblot analysis using antibodies against OK1, AGPase (plastidic
    Figure Legend Snippet: OK1 is located in chloroplasts. Equal amounts of protein extracted from Arabidopsis leaf protoplasts (P) and chloroplasts (C), respectively, were separated by SDS-PAGE and examined by immunoblot analysis using antibodies against OK1, AGPase (plastidic

    Techniques Used: SDS Page

    In vitro activity assay with recombinant OK1 protein. A, OK1 phosphorylates phosphoglucans but not phosphate free glucans. One microgram recombinant OK1 protein was incubated for 15 min with 25 μ m ATP containing 0.5 μ Ci [ βγ
    Figure Legend Snippet: In vitro activity assay with recombinant OK1 protein. A, OK1 phosphorylates phosphoglucans but not phosphate free glucans. One microgram recombinant OK1 protein was incubated for 15 min with 25 μ m ATP containing 0.5 μ Ci [ βγ

    Techniques Used: In Vitro, Activity Assay, Recombinant, Incubation

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.338426

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

    Article Snippet: Two types of 219-bp genomic fragments (G/G and C/C) of the 3′-UTR of TLR4 at SNP rs11536889 were amplified from two different human genomic DNAs with the Expand High Fidelity PCR system using the primer sets (5′-TGG GAT CCC TCC CCT GTA CCC TTC-3′ (sense) and 5′-CTG GAT CCG TTT CTG AGG AGG CTG GAT G-3′ (antisense)).

    Techniques: Expressing, Isolation, Quantitative RT-PCR

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkl852

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

    Article Snippet: Plasmid construction DNA inserts for plasmid constructs were amplified by PCR using the Expand High Fidelity PCR system (Roche) and primers linked to restriction enzyme sites suitable for cloning.

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkl852

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

    Article Snippet: Plasmid construction DNA inserts for plasmid constructs were amplified by PCR using the Expand High Fidelity PCR system (Roche) and primers linked to restriction enzyme sites suitable for cloning.

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

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

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

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

    doi:

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

    Article Snippet: PCR for MSS HTA was done by using the Expand High Fidelity PCR System (Roche Molecular Biochemicals) with the following modifications: reactions contained 0.2–4 μg of total cellular DNA, 1× Titan RT-PCR buffer, 3 mM MgCl2 , 0.2 mM of each dNTP, 5 mM DTT, 0.5 μM primers PRAMPUP and PRAMPDW, and 1 μl of Expand enzyme mix.

    Techniques: Polymerase Chain Reaction, Amplification

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

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

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

    doi:

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

    Article Snippet: PCR for MSS HTA was done by using the Expand High Fidelity PCR System (Roche Molecular Biochemicals) with the following modifications: reactions contained 0.2–4 μg of total cellular DNA, 1× Titan RT-PCR buffer, 3 mM MgCl2 , 0.2 mM of each dNTP, 5 mM DTT, 0.5 μM primers PRAMPUP and PRAMPDW, and 1 μl of Expand enzyme mix.

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

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

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

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

    doi:

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

    Article Snippet: PCR for MSS HTA was done by using the Expand High Fidelity PCR System (Roche Molecular Biochemicals) with the following modifications: reactions contained 0.2–4 μg of total cellular DNA, 1× Titan RT-PCR buffer, 3 mM MgCl2 , 0.2 mM of each dNTP, 5 mM DTT, 0.5 μM primers PRAMPUP and PRAMPDW, and 1 μl of Expand enzyme mix.

    Techniques: Labeling, Polymerase Chain Reaction

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

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

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

    doi:

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

    Article Snippet: PCR for MSS HTA was done by using the Expand High Fidelity PCR System (Roche Molecular Biochemicals) with the following modifications: reactions contained 0.2–4 μg of total cellular DNA, 1× Titan RT-PCR buffer, 3 mM MgCl2 , 0.2 mM of each dNTP, 5 mM DTT, 0.5 μM primers PRAMPUP and PRAMPDW, and 1 μl of Expand enzyme mix.

    Techniques: Labeling, Polymerase Chain Reaction, Mutagenesis

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

    Journal: Infection and Immunity

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

    doi: 10.1128/IAI.70.6.3234-3248.2002

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

    Article Snippet: 1 μg of total RNA in the presence of random decamers according to the conditions specified by the manufacturer. cDNAs were amplified by PCR by using the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis, Ind.) in a 50-μl reaction mixture containing 1× Expand HF buffer, 1.5 mM MgCl2 , 200 μM (each) deoxynucleoside triphosphates, 1 μM forward and reverse primers, and 1.5 U of Taq polymerase.

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