Structured Review

Thermo Fisher xopq
Virulence deficiency associated with xopN - , <t>xopQ</t> - , <t>xopX</t> - or xopZ - single mutants of Xanthomonas oryzae pv. oryzae . Leaves of susceptible rice variety Taichung-Native 1 (TN-1) were clip inoculated with the following X . oryzae pv. oryzae strains: ( A ) wild type, xopN - mutant, xopN- / xopN + (complemented strain), xopN - /pHM1 (vector control), xopX - mutant, xopX- / xopX + (complemented strain), xopX - /pHM1 (vector control) or xopN - xopX - double mutant strains; ( B ) wild type, xopQ - mutant, xopQ- / xopQ + (complemented strain), xopQ - /pHM1 (vector control), xopZ - mutant, xopZ- / xopZ + (complemented strain) or xopZ - /pHM1 (vector control). Lesion lengths were measured 7 days post inoculation. Error bars indicate the standard deviation of readings from at least 10 inoculated leaves. Similar results were obtained in independent experiments. A Student’s two-tai led t test for independent means was performed for the following values: wild type with each of the single mutants and the double mutant with each of the single mutants with correction for multiple comparisons, mutant with empty vector and complemented strains for each single mutant. The brackets on the graphs indicate the comparisons that were made. All compared values are significantly different at P
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Images

1) Product Images from "Cell Wall Degrading Enzyme Induced Rice Innate Immune Responses Are Suppressed by the Type 3 Secretion System Effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae"

Article Title: Cell Wall Degrading Enzyme Induced Rice Innate Immune Responses Are Suppressed by the Type 3 Secretion System Effectors XopN, XopQ, XopX and XopZ of Xanthomonas oryzae pv. oryzae

Journal: PLoS ONE

doi: 10.1371/journal.pone.0075867

Virulence deficiency associated with xopN - , xopQ - , xopX - or xopZ - single mutants of Xanthomonas oryzae pv. oryzae . Leaves of susceptible rice variety Taichung-Native 1 (TN-1) were clip inoculated with the following X . oryzae pv. oryzae strains: ( A ) wild type, xopN - mutant, xopN- / xopN + (complemented strain), xopN - /pHM1 (vector control), xopX - mutant, xopX- / xopX + (complemented strain), xopX - /pHM1 (vector control) or xopN - xopX - double mutant strains; ( B ) wild type, xopQ - mutant, xopQ- / xopQ + (complemented strain), xopQ - /pHM1 (vector control), xopZ - mutant, xopZ- / xopZ + (complemented strain) or xopZ - /pHM1 (vector control). Lesion lengths were measured 7 days post inoculation. Error bars indicate the standard deviation of readings from at least 10 inoculated leaves. Similar results were obtained in independent experiments. A Student’s two-tai led t test for independent means was performed for the following values: wild type with each of the single mutants and the double mutant with each of the single mutants with correction for multiple comparisons, mutant with empty vector and complemented strains for each single mutant. The brackets on the graphs indicate the comparisons that were made. All compared values are significantly different at P
Figure Legend Snippet: Virulence deficiency associated with xopN - , xopQ - , xopX - or xopZ - single mutants of Xanthomonas oryzae pv. oryzae . Leaves of susceptible rice variety Taichung-Native 1 (TN-1) were clip inoculated with the following X . oryzae pv. oryzae strains: ( A ) wild type, xopN - mutant, xopN- / xopN + (complemented strain), xopN - /pHM1 (vector control), xopX - mutant, xopX- / xopX + (complemented strain), xopX - /pHM1 (vector control) or xopN - xopX - double mutant strains; ( B ) wild type, xopQ - mutant, xopQ- / xopQ + (complemented strain), xopQ - /pHM1 (vector control), xopZ - mutant, xopZ- / xopZ + (complemented strain) or xopZ - /pHM1 (vector control). Lesion lengths were measured 7 days post inoculation. Error bars indicate the standard deviation of readings from at least 10 inoculated leaves. Similar results were obtained in independent experiments. A Student’s two-tai led t test for independent means was performed for the following values: wild type with each of the single mutants and the double mutant with each of the single mutants with correction for multiple comparisons, mutant with empty vector and complemented strains for each single mutant. The brackets on the graphs indicate the comparisons that were made. All compared values are significantly different at P

Techniques Used: Cross-linking Immunoprecipitation, Mutagenesis, Plasmid Preparation, Standard Deviation

Agrobacterium mediated transient transfer of xopQ , xopX and xopZ genes of Xanthomonas oryzae pv. oryzae suppresses LipA induced programmed cell death in rice roots. Rice roots were treated with the following: buffer + Estradiol (Est; A ); LipA + Est ( B ); pretreatment with EHA105/pMDC7- xopQ with Est ( C ); EHA105/pMDC7- xopX with Est ( E ) and EHA105/pMDC7- xopZ with Est ( G ) followed by treatment with LipA and Est ( C , E and G ); pretreatment with EHA105/pMDC7- xopQ ( D ); EHA105/pMDC7- xopX ( F ) and EHA105/pMDC7- xopZ ( H ) followed by treatment with LipA ( D , F and H ). The roots were subsequently stained with propidium iodide (PI) and visualized under a confocal microscope. Dispersed intracellular PI staining is indicative of programmed cell death in rice roots. Scale bar measures 20µm.
Figure Legend Snippet: Agrobacterium mediated transient transfer of xopQ , xopX and xopZ genes of Xanthomonas oryzae pv. oryzae suppresses LipA induced programmed cell death in rice roots. Rice roots were treated with the following: buffer + Estradiol (Est; A ); LipA + Est ( B ); pretreatment with EHA105/pMDC7- xopQ with Est ( C ); EHA105/pMDC7- xopX with Est ( E ) and EHA105/pMDC7- xopZ with Est ( G ) followed by treatment with LipA and Est ( C , E and G ); pretreatment with EHA105/pMDC7- xopQ ( D ); EHA105/pMDC7- xopX ( F ) and EHA105/pMDC7- xopZ ( H ) followed by treatment with LipA ( D , F and H ). The roots were subsequently stained with propidium iodide (PI) and visualized under a confocal microscope. Dispersed intracellular PI staining is indicative of programmed cell death in rice roots. Scale bar measures 20µm.

Techniques Used: Staining, Microscopy

A xopN - xopQ - xopX - xopZ - quadruple mutant of Xanthomonas oryzae pv. oryzae induces callose deposition in rice leaves. Rice leaves were infiltrated with one of the following: water ( A ); wild type Xoo ( B ); xopN - xopQ - xopX - xopZ - quadruple mutant ( C ) and T3S - mutant ( D ). The treated leaves were subsequently stained with aniline blue and visualized under an epifluorescence microscope. White dots in these pictures are indicative of callose deposition. Scale bar measures 100µm. ( E ) Mean and standard deviation were calculated for number of callose deposits observed in a leaf area of 0.60 mm 2 . Data were collected from at least five leaves per treatment in each experiment (three experiments indicated as ExpI, ExpII and ExpIII) and two to three different viewing areas from the infiltrated region of each leaf. Statistically significant differences at P
Figure Legend Snippet: A xopN - xopQ - xopX - xopZ - quadruple mutant of Xanthomonas oryzae pv. oryzae induces callose deposition in rice leaves. Rice leaves were infiltrated with one of the following: water ( A ); wild type Xoo ( B ); xopN - xopQ - xopX - xopZ - quadruple mutant ( C ) and T3S - mutant ( D ). The treated leaves were subsequently stained with aniline blue and visualized under an epifluorescence microscope. White dots in these pictures are indicative of callose deposition. Scale bar measures 100µm. ( E ) Mean and standard deviation were calculated for number of callose deposits observed in a leaf area of 0.60 mm 2 . Data were collected from at least five leaves per treatment in each experiment (three experiments indicated as ExpI, ExpII and ExpIII) and two to three different viewing areas from the infiltrated region of each leaf. Statistically significant differences at P

Techniques Used: Mutagenesis, Staining, Microscopy, Standard Deviation

Agrobacterium mediated transient transfer of xopQ , xopX and xopZ genes of Xanthomonas oryzae pv. oryzae suppresses LipA induced callose deposition in rice leaves. Rice leaves were infiltrated with one of the following: buffer + Estradiol (Est; A ); LipA + Est ( B ); EHA105/pMDC7- xopQ + LipA with Est ( C ) or without Est ( D ); EHA105/pMDC7- xopX + LipA with Est ( E ) or without Est ( F ); EHA105/pMDC7- xopZ + LipA with Est ( G ) or without Est ( H ). The leaves were subsequently stained with aniline blue and visualized under an epifluorescence microscope. Callose deposition is seen as white spots in these pictures. Scale bar measures 100µm. ( I ) Mean and standard deviation were calculated for number of callose deposits observed in an area of 0.60 mm 2 . Data were collected from at least five leaves per treatment in each experiment (three experiments indicated as ExpI, ExpII and ExpIII) and two to three different viewing areas from the infiltrated region of each leaf. Statistically significant differences at P
Figure Legend Snippet: Agrobacterium mediated transient transfer of xopQ , xopX and xopZ genes of Xanthomonas oryzae pv. oryzae suppresses LipA induced callose deposition in rice leaves. Rice leaves were infiltrated with one of the following: buffer + Estradiol (Est; A ); LipA + Est ( B ); EHA105/pMDC7- xopQ + LipA with Est ( C ) or without Est ( D ); EHA105/pMDC7- xopX + LipA with Est ( E ) or without Est ( F ); EHA105/pMDC7- xopZ + LipA with Est ( G ) or without Est ( H ). The leaves were subsequently stained with aniline blue and visualized under an epifluorescence microscope. Callose deposition is seen as white spots in these pictures. Scale bar measures 100µm. ( I ) Mean and standard deviation were calculated for number of callose deposits observed in an area of 0.60 mm 2 . Data were collected from at least five leaves per treatment in each experiment (three experiments indicated as ExpI, ExpII and ExpIII) and two to three different viewing areas from the infiltrated region of each leaf. Statistically significant differences at P

Techniques Used: Staining, Microscopy, Standard Deviation

2) Product Images from "Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes"

Article Title: Expression of dedifferentiation markers and multilineage markers in U251 glioblastoma cells with silenced EGFR and FGFR genes

Journal: Oncology Letters

doi: 10.3892/ol.2013.1685

Identification of EGFP-labeled lentiviral expression vector. (A-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-EGFR-miR. M, marker DL2000; lane 1, negative control; lanes 2–4, PCR product of recombinant vector (911 bp). (A-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-EGFR-miR. M, marker DL15000; lane 1, digestion product of double restriction endonuclease of Asc I and Pme I; lane 2, the recombinant plasmid pLenti6.3-EGFR-EGFR-miR. (B-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, negative control; lanes 2–6, PCR product of recombinant vector (911 bp). (B-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, digestion product of double restriction endonuclease of AscI and PmeI; lane 2, the recombinant plasmid pLenti6.3-EGFP-FGFR-miR. EGFP, enhanced green fluorescent protein; EGFR, epithelial growth factor receptor; miR, microRNA; PCR, polymerase chain reaction; FGFR, fibroblast growth factor receptor.
Figure Legend Snippet: Identification of EGFP-labeled lentiviral expression vector. (A-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-EGFR-miR. M, marker DL2000; lane 1, negative control; lanes 2–4, PCR product of recombinant vector (911 bp). (A-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-EGFR-miR. M, marker DL15000; lane 1, digestion product of double restriction endonuclease of Asc I and Pme I; lane 2, the recombinant plasmid pLenti6.3-EGFR-EGFR-miR. (B-1) Gel electrophoresis of the PCR product of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, negative control; lanes 2–6, PCR product of recombinant vector (911 bp). (B-2) Restriction map of the PCR positive vector of pLenti6.3-EGFP-FGFR-miR. M, marker DL5000; lane 1, digestion product of double restriction endonuclease of AscI and PmeI; lane 2, the recombinant plasmid pLenti6.3-EGFP-FGFR-miR. EGFP, enhanced green fluorescent protein; EGFR, epithelial growth factor receptor; miR, microRNA; PCR, polymerase chain reaction; FGFR, fibroblast growth factor receptor.

Techniques Used: Labeling, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Marker, Negative Control, Recombinant

3) Product Images from "Nuclear Translocation of Acinetobacter baumannii Transposase Induces DNA Methylation of CpG Regions in the Promoters of E-cadherin Gene"

Article Title: Nuclear Translocation of Acinetobacter baumannii Transposase Induces DNA Methylation of CpG Regions in the Promoters of E-cadherin Gene

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038974

A. baumannii transposase targets in the nucleus of host cells via NLSs. COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp 1–362 and Tnp 1–230 , were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp 1–37 and Tnp 1–224 , were located in the cytoplasm.
Figure Legend Snippet: A. baumannii transposase targets in the nucleus of host cells via NLSs. COS-7 cells were transfected with the plasmid constructs of transposase gene cloned in the destination vector pcDNATM6.2/N-EmGFP-DEST and incubated for 24 h. The subcellular localization of transposase proteins fused with GFP was observed by confocal laser microscopy. Two A. baumannii transposase proteins with NLSs, Tnp 1–362 and Tnp 1–230 , were located in the nuclei of host cells, whereas transposase proteins without NLSs, Tnp 1–37 and Tnp 1–224 , were located in the cytoplasm.

Techniques Used: Transfection, Plasmid Preparation, Construct, Clone Assay, Incubation, Microscopy

Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene. (A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods . Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp 1–37 ; 7, A549 cells transfected with plasmid constructs of Tnp 1–224 ; 8, A549 cells transfected with plasmid constructs of Tnp 1–230 ; 9, A549 cells transfected with plasmid constructs of Tnp 1–362 . (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods . Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p
Figure Legend Snippet: Nuclear targeting of A. baumannii transposase specifically induces DNA methylation in CpG regions and down-regulates expression of E-cadherin gene. (A) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. The genomic DNA was purified and methylation-specific PCR with methylated and unmethylated primers was performed as described in materials and methods . Lane 1, molecular size marker; 2, unmethylated DNA; 3, methylated DNA; 4, A549 cells; 5, A549 cells transfected with the destination vector pcDNA™6.2/N-EmGFP-DEST; 6, A549 cells transfected with plasmid constructs of Tnp 1–37 ; 7, A549 cells transfected with plasmid constructs of Tnp 1–224 ; 8, A549 cells transfected with plasmid constructs of Tnp 1–230 ; 9, A549 cells transfected with plasmid constructs of Tnp 1–362 . (B) A549 cells were transfected with A. baumannii transposase clones and incubated for 48 h. Total RNA was extracted and qRT-PCR was performed as described in materials and methods . Data are presented as mean ± SD of triplicate determinations. Asterisks indicate a statistically significant difference between A549 cells transfected with the empty destination vector and plasmid constructs of A. baumannii transposase fused with GFP (student's t-test p

Techniques Used: DNA Methylation Assay, Expressing, Transfection, Clone Assay, Incubation, Purification, Methylation, Polymerase Chain Reaction, Marker, Plasmid Preparation, Construct, Quantitative RT-PCR

4) Product Images from "Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes"

Article Title: Promoter analysis of macrophage- and tick cell-specific differentially expressed Ehrlichia chaffeensis p28-Omp genes

Journal: BMC Microbiology

doi: 10.1186/1471-2180-9-99

(A) Green fluorescent protein (GFP) constructs evaluated for the promoter activity of p28-Omp genes 14 and 19 . The pPROBE-NT plasmids containing the promoterless GFP gene (2 and 3) and upstream sequences of genes 14 and 19 in front of the GFP gene (1 and 4, respectively) and a construct containing no promoter sequence were evaluated for GFP expression in E. coli . (B) LacZ constructs evaluated for the promoter activity of p28-Omp genes 14 and 19. The pBlue-TOPO vector containing promoterless lacZ gene (pBlue-TOPO) and upstream sequences of genes 14 and 19 inserted in forward (14-F and 19-F) and reverse orientations (14-R and 19-R) were evaluated for β-galactosidase activity in E. coli . Data are presented with SD values calculated from four independent experiments ( P ≤ 0.001).
Figure Legend Snippet: (A) Green fluorescent protein (GFP) constructs evaluated for the promoter activity of p28-Omp genes 14 and 19 . The pPROBE-NT plasmids containing the promoterless GFP gene (2 and 3) and upstream sequences of genes 14 and 19 in front of the GFP gene (1 and 4, respectively) and a construct containing no promoter sequence were evaluated for GFP expression in E. coli . (B) LacZ constructs evaluated for the promoter activity of p28-Omp genes 14 and 19. The pBlue-TOPO vector containing promoterless lacZ gene (pBlue-TOPO) and upstream sequences of genes 14 and 19 inserted in forward (14-F and 19-F) and reverse orientations (14-R and 19-R) were evaluated for β-galactosidase activity in E. coli . Data are presented with SD values calculated from four independent experiments ( P ≤ 0.001).

Techniques Used: Construct, Activity Assay, Sequencing, Expressing, Plasmid Preparation

5) Product Images from "STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells"

Article Title: STAT5 regulation of BCL10 parallels constitutive NF?B activation in lymphoid tumor cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-8-67

(A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents
Figure Legend Snippet: (A) Generation of a library encoding STAT5-responsive genomic elements by ChIP-cloning . IL-2 stimulated, formaldehyde cross-linked YT cells were lysed, sonicated and immuno-precipitated with antibodies to STAT5A or STAT5B. Eluted DNA was ligated to a unidirectional linker (black blocks), amplified and then cloned into pCR II-TOPO vector. Clones containing inserts were identified by sequencing. (B) Successful immuno-precipitation of STAT5 from formaldehyde fixed YT cell lysates . YT cells were stimulated with medium (-) or IL-2 (+) then fixed with formaldehyde. Fixed lysates were immuno-precipitated with antibodies to STAT5 as indicated or normal rabbit serum (IgG CTRL) then Western blotted for STAT5. Molecular weight markers are indicated to the left side of the panel. Input material corresponds to 1% of cell lysate used in the immuno-precipitations. (C) Validation of STAT5 ChIP in YT cells . ChIP assay with C-terminal antibodies to STAT5A and B in combination (αSTAT5 C-term) or IgG control was carried out as described above. The eluted DNA was then used as template in qPCR reactions with primers designed to PRR III. (D) STAT5 bound genomic library captured by ChIP-cloning . Inserts were amplified via PCR using M13 primers prior to sequencing and visualized by agarose gel electrophoresis (1%). Stars (*) indicate clones without an insert. (E) Nearby gene mapping of the ChIP-clone identified genomic sequences . One hundred and nineteen clones were sequenced, 3 fragments were duplicates and 9 were greater than 300 kb away from any coding region. The remaining sequences that fell within 300 kb from coding regions were analyzed with Cis-Regulatory Element Annotation System (CEAS). The pie chart represents "%" distribution.

Techniques Used: Chromatin Immunoprecipitation, Clone Assay, Sonication, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Immunoprecipitation, Western Blot, Molecular Weight, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Genomic Sequencing

6) Product Images from "HDAC Inhibitors Repress BARD1 Isoform Expression in Acute Myeloid Leukemia Cells via Activation of miR-19a and/or b"

Article Title: HDAC Inhibitors Repress BARD1 Isoform Expression in Acute Myeloid Leukemia Cells via Activation of miR-19a and/or b

Journal: PLoS ONE

doi: 10.1371/journal.pone.0083018

BARD1 is the target of miR-19a and miR-19b. ( A ) Real-Time PCR for mir-19a and miR-19b in NB4 cells transfected with mimic-miR-19a, mimic-miR-19b or mimic-miR-scramble (miR-s). Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( B ) BARD1 Real-Time PCR in NB4 cells transfected with mimic-miR-19a, mimic-miR-19b or mimic-miR-scramble. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( C ) Luciferase assay in HeLa cells after transfection with 1 µg pGL3-3’UTR-BARD1 wild-type (left) and mutated (right) vectors plus mimic-miR-19a/b or scramble at a concentration of 200 nM. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. Annealing of miR-19a and miR-19b to BARD1 3’UTR: wild type and mutated BARD1 3’UTR are schematized.
Figure Legend Snippet: BARD1 is the target of miR-19a and miR-19b. ( A ) Real-Time PCR for mir-19a and miR-19b in NB4 cells transfected with mimic-miR-19a, mimic-miR-19b or mimic-miR-scramble (miR-s). Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( B ) BARD1 Real-Time PCR in NB4 cells transfected with mimic-miR-19a, mimic-miR-19b or mimic-miR-scramble. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( C ) Luciferase assay in HeLa cells after transfection with 1 µg pGL3-3’UTR-BARD1 wild-type (left) and mutated (right) vectors plus mimic-miR-19a/b or scramble at a concentration of 200 nM. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. Annealing of miR-19a and miR-19b to BARD1 3’UTR: wild type and mutated BARD1 3’UTR are schematized.

Techniques Used: Real-time Polymerase Chain Reaction, Transfection, Luciferase, Concentration Assay

Vorinostat induces overexpression of different miRNAs in human AML cell lines. ( A ) Heat-map of miRNAs altered in NB4 cells after treatment with Vorinostat (5 µM) for 6 h (s); the experiment was performed in biological triplicate and the t-test analysis was carried out between NB4 untreated (NB NT 1-3) and NB4 treated with Vorinostat (NB 6h s1-3); p-value ≤ 0.10. ( B ) Table of miRNAs up-regulated (in red) and down-regulated (in green) after Vorinostat treatment; p-value ≤ 0.10. ( C ) Annealing of miR-19a and miR-19b to BARD1 3’UTR. ( D ) Real-Time PCR for miR-19a and miR-19b in NB4, K562, U937 and HL60 cells after Vorinostat at a concentration of 5 µM. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column.
Figure Legend Snippet: Vorinostat induces overexpression of different miRNAs in human AML cell lines. ( A ) Heat-map of miRNAs altered in NB4 cells after treatment with Vorinostat (5 µM) for 6 h (s); the experiment was performed in biological triplicate and the t-test analysis was carried out between NB4 untreated (NB NT 1-3) and NB4 treated with Vorinostat (NB 6h s1-3); p-value ≤ 0.10. ( B ) Table of miRNAs up-regulated (in red) and down-regulated (in green) after Vorinostat treatment; p-value ≤ 0.10. ( C ) Annealing of miR-19a and miR-19b to BARD1 3’UTR. ( D ) Real-Time PCR for miR-19a and miR-19b in NB4, K562, U937 and HL60 cells after Vorinostat at a concentration of 5 µM. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column.

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Concentration Assay

Modulation of miR-19a, miR-19b and BARD1increases mortality of U937 cells after Vorinostat treatment. ( A ) Cell death analysis in U937-mir cells after 24 h treatment with Vorinostat (5 µM). Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. (B) Caspase-8 and -9 activation in U937-mir cells treated for 24 h with Vorinostat (5 µM). Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( C ) BARD1 expression levels measured by Real-Time PCR in U937 cells transfected with specific siRNAs as indicated. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( D ) Analysis of cell death by PI incorporation after 24 h of Vorinostat treatment (5 µM) in U937 siRNA-transfected cells. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column.
Figure Legend Snippet: Modulation of miR-19a, miR-19b and BARD1increases mortality of U937 cells after Vorinostat treatment. ( A ) Cell death analysis in U937-mir cells after 24 h treatment with Vorinostat (5 µM). Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. (B) Caspase-8 and -9 activation in U937-mir cells treated for 24 h with Vorinostat (5 µM). Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( C ) BARD1 expression levels measured by Real-Time PCR in U937 cells transfected with specific siRNAs as indicated. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column. ( D ) Analysis of cell death by PI incorporation after 24 h of Vorinostat treatment (5 µM) in U937 siRNA-transfected cells. Data show the mean value of three parallel experiments with error bars showing the standard deviations on top of each column.

Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection

7) Product Images from "Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing"

Article Title: Grafting on a Non-Transgenic Tolerant Tomato Variety Confers Resistance to the Infection of a Sw5-Breaking Strain of Tomato spotted wilt virus via RNA Silencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141319

VIGS of RDR1 and RDR6 in Sl-Ma and Sl-UC plants. Relative quantity (RQ) of RDR1 and RDR6 transcripts (columns) in samples of Sl - Ma and Sl - UC plants collected at 14 dpa with A . tumefaciens carrying pTRV1+ pTRV2-RDR1 (Sl-Ma TRV-R1 and Sl-UC TRV-R6) and pTRV1+ pTRV2-RDR6 (Sl-Ma TRV-R1 and Sl-Ma TRV-R6). RQ values were first normalized on the accumulation level of the GAPDH mRNA (Δ cycle threshold [Ct] = Ct GAPDH –Ct target RNA) and then used to determine the relative quantification of each target RNA with a calibrator, according to the formula ΔΔCt = ΔCt calibrator –ΔCt target RNA. Each target mRNA in an individual mock-inoculated plant served as calibrator (RQ set to 1) for the respective gene. RQ for RDR1 and RDR6 transcripts was deduced by the formula expression 2 -ΔΔCt . Columns represent mean RQ values from three biological replicates and different letters represent statistically significant differences values according to separate one-way ANOVA analysis for each target mRNA, using Tukey’s test (P = 0.05). Vertical bars on columns represent standard deviations among replicates. Figure shows also estimates of the accumulation of TSWV-CiPz RNA (red line) in agroinfiltrated plants. After collection of leaf samples, plants were inoculated with TSWV-CiPZ on the first and second true leaves above remnants of cotyledons and load of viral RNA estimated at 19 dpi with dot blot hybridization. Vertical bars on line represent standard deviations among replicates.
Figure Legend Snippet: VIGS of RDR1 and RDR6 in Sl-Ma and Sl-UC plants. Relative quantity (RQ) of RDR1 and RDR6 transcripts (columns) in samples of Sl - Ma and Sl - UC plants collected at 14 dpa with A . tumefaciens carrying pTRV1+ pTRV2-RDR1 (Sl-Ma TRV-R1 and Sl-UC TRV-R6) and pTRV1+ pTRV2-RDR6 (Sl-Ma TRV-R1 and Sl-Ma TRV-R6). RQ values were first normalized on the accumulation level of the GAPDH mRNA (Δ cycle threshold [Ct] = Ct GAPDH –Ct target RNA) and then used to determine the relative quantification of each target RNA with a calibrator, according to the formula ΔΔCt = ΔCt calibrator –ΔCt target RNA. Each target mRNA in an individual mock-inoculated plant served as calibrator (RQ set to 1) for the respective gene. RQ for RDR1 and RDR6 transcripts was deduced by the formula expression 2 -ΔΔCt . Columns represent mean RQ values from three biological replicates and different letters represent statistically significant differences values according to separate one-way ANOVA analysis for each target mRNA, using Tukey’s test (P = 0.05). Vertical bars on columns represent standard deviations among replicates. Figure shows also estimates of the accumulation of TSWV-CiPz RNA (red line) in agroinfiltrated plants. After collection of leaf samples, plants were inoculated with TSWV-CiPZ on the first and second true leaves above remnants of cotyledons and load of viral RNA estimated at 19 dpi with dot blot hybridization. Vertical bars on line represent standard deviations among replicates.

Techniques Used: Expressing, Dot Blot, Hybridization

8) Product Images from "Lnc-CC3 increases metastasis in cervical cancer by increasing Slug expression"

Article Title: Lnc-CC3 increases metastasis in cervical cancer by increasing Slug expression

Journal: Oncotarget

doi: 10.18632/oncotarget.9519

Lnc-CC3 enhanced the lung colonization capacity of SiHa cells ( A ) Representative images of nude mouse lungs; arrows indicate clusters of nodules on the lung surface. ( B ) Metastatic tumor nodules on the lung surface were counted under stereomicroscope. Each group contained 6 nude mice. ( C ) Hematoxylin-eosin staining of lung tumor nodules. Photographed at 100× magnification, scale bar = 400 μm. ( D ) Neoplasms were found in all pathological sections from lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SEM of independent experiments ( n = 6), * p
Figure Legend Snippet: Lnc-CC3 enhanced the lung colonization capacity of SiHa cells ( A ) Representative images of nude mouse lungs; arrows indicate clusters of nodules on the lung surface. ( B ) Metastatic tumor nodules on the lung surface were counted under stereomicroscope. Each group contained 6 nude mice. ( C ) Hematoxylin-eosin staining of lung tumor nodules. Photographed at 100× magnification, scale bar = 400 μm. ( D ) Neoplasms were found in all pathological sections from lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SEM of independent experiments ( n = 6), * p

Techniques Used: Mouse Assay, Staining

lnc-CC3 full length clone and sequence analysis ( A ) Electrophoresis of the RACE product. ( B ) The full sequence of lnc-CC3. ( C ) The chromosomal location of lnc-CC3 was determined using NCBI MapViewer; the red label indicates its position. ( D ) Northern blot results for lnc-CC3. ( E ) In situ hybridization of lnc-CC3 in SiHa cells; the photograph was taken at 200× magnification. ( F ) Putative proteins encoded by lnc-CC3 as predicted by ORF Finder; predicted proteins were subject to Blastp. ( G ) Conservation analysis of lnc-CC3 was conducted using Clustal Omega; repeat sequence analysis was conducted using RepeatMasker.
Figure Legend Snippet: lnc-CC3 full length clone and sequence analysis ( A ) Electrophoresis of the RACE product. ( B ) The full sequence of lnc-CC3. ( C ) The chromosomal location of lnc-CC3 was determined using NCBI MapViewer; the red label indicates its position. ( D ) Northern blot results for lnc-CC3. ( E ) In situ hybridization of lnc-CC3 in SiHa cells; the photograph was taken at 200× magnification. ( F ) Putative proteins encoded by lnc-CC3 as predicted by ORF Finder; predicted proteins were subject to Blastp. ( G ) Conservation analysis of lnc-CC3 was conducted using Clustal Omega; repeat sequence analysis was conducted using RepeatMasker.

Techniques Used: Sequencing, Electrophoresis, Northern Blot, In Situ Hybridization

Lnc-CC3 over-expression increased migration and invasion in SiHa cells in vitro ( A ) In situ hybridization results for the cervical tissue microarray revealed that lnc-CC3 is mainly localized in the cytoplasm. Normal, normal cervical tissue; CC _ Negative, cervical cancer negative; CC _ Positive, cervical cancer positive. 200× magnification, scale bar = 200 μm. ( B ) Lnc-CC3 levels were elevated more frequently in stage III cervical cancer. Normal, normal cervical tissue; I, II, III represent the different cervical cancer stages. Statistical significance was determined using the χ 2 test, p = 0.003. ( C ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( D ) Cell proliferation curve for SiHa cells determined using the CCK-8 assay ( n = 3). ( E ) Cell cycle distribution of SiHa cells was determined by flow cytometry ( n = 3). ( F ) Colony formation assay results for SiHa cells ( n = 3). ( G ) Cell migration capacity was determined using a wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification at the same location in each well. ( H ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Untransfected and pcDNA3.1 (+) plasmid transfected SiHa cell lines were used as controls; lnc-CC3 indicates lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SD of independent experiments, * p
Figure Legend Snippet: Lnc-CC3 over-expression increased migration and invasion in SiHa cells in vitro ( A ) In situ hybridization results for the cervical tissue microarray revealed that lnc-CC3 is mainly localized in the cytoplasm. Normal, normal cervical tissue; CC _ Negative, cervical cancer negative; CC _ Positive, cervical cancer positive. 200× magnification, scale bar = 200 μm. ( B ) Lnc-CC3 levels were elevated more frequently in stage III cervical cancer. Normal, normal cervical tissue; I, II, III represent the different cervical cancer stages. Statistical significance was determined using the χ 2 test, p = 0.003. ( C ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( D ) Cell proliferation curve for SiHa cells determined using the CCK-8 assay ( n = 3). ( E ) Cell cycle distribution of SiHa cells was determined by flow cytometry ( n = 3). ( F ) Colony formation assay results for SiHa cells ( n = 3). ( G ) Cell migration capacity was determined using a wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification at the same location in each well. ( H ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Untransfected and pcDNA3.1 (+) plasmid transfected SiHa cell lines were used as controls; lnc-CC3 indicates lnc-CC3 overexpressing SiHa cells. Data are expressed as mean ± SD of independent experiments, * p

Techniques Used: Over Expression, Migration, In Vitro, In Situ Hybridization, Microarray, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Cytometry, Colony Assay, Wound Healing Assay, Transwell Assay, Plasmid Preparation, Transfection

Lnc-CC3 knockdown suppressed migration and invasion in SiHa cells in vitro ( A ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( B ) The shape of lnc-CC3 knockdown SiHa cells changed from elongated to round. Photograph at 100× magnification, scale bar = 200 μm. ( C ) Cell migration capacity was determined by wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification, at the same location in each well. ( D ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Data are expressed as mean ± SD of independent experiments, * p
Figure Legend Snippet: Lnc-CC3 knockdown suppressed migration and invasion in SiHa cells in vitro ( A ) Fold change in lnc-CC3 expression in SiHa cells was analyzed by qRT-PCR ( n = 3); β-actin was used as an internal control. Data were analyzed using the 2 −ΔΔCT method. ( B ) The shape of lnc-CC3 knockdown SiHa cells changed from elongated to round. Photograph at 100× magnification, scale bar = 200 μm. ( C ) Cell migration capacity was determined by wound healing assay ( n = 3). Photographs were taken immediately at 0 h and 48 h after wounding at 100× magnification, at the same location in each well. ( D ) Representative images of the migration and invasion transwell assay; invaded cell number was determined by photograph at 200× magnification in five random views per chamber. Data are expressed as mean ± SD of independent experiments, * p

Techniques Used: Migration, In Vitro, Expressing, Quantitative RT-PCR, Wound Healing Assay, Transwell Assay

Lnc-CC3 promoted EMT in cervical cancer cells by increasing Slug expression ( A ) Lnc-CC3 overexpression increased the expression of Slug and Snail, and lnc-CC3 knockdown increased the expression of E-cadherin and β-catenin. ( B ) Changes in Slug, Snail, E-cadherin, and β-catenin protein levels in SiHa cells. The effects of lnc-CC3 overexpression on Snail and Slug mRNA ( C ) and protein ( D ) levels in CaSki and HeLa cells. The qRT-PCR data were analyzed using the 2 −ΔΔCT method and WB data were quantified by densitometry using ImageJ software. Data are expressed as mean ± SD of independent experiments ( n = 3), * p
Figure Legend Snippet: Lnc-CC3 promoted EMT in cervical cancer cells by increasing Slug expression ( A ) Lnc-CC3 overexpression increased the expression of Slug and Snail, and lnc-CC3 knockdown increased the expression of E-cadherin and β-catenin. ( B ) Changes in Slug, Snail, E-cadherin, and β-catenin protein levels in SiHa cells. The effects of lnc-CC3 overexpression on Snail and Slug mRNA ( C ) and protein ( D ) levels in CaSki and HeLa cells. The qRT-PCR data were analyzed using the 2 −ΔΔCT method and WB data were quantified by densitometry using ImageJ software. Data are expressed as mean ± SD of independent experiments ( n = 3), * p

Techniques Used: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Software

Screening for new genes associated with cervical cancer ( A ) Genbank ID and description of the 7 identified ESTs. ( B ) PCR showed that CC3 was present as a single band in the gel. ( C ) CC3 was expressed in cervical cancer cell lines; expression was higher in SiHa cells than in HeLa and CaSki cells.
Figure Legend Snippet: Screening for new genes associated with cervical cancer ( A ) Genbank ID and description of the 7 identified ESTs. ( B ) PCR showed that CC3 was present as a single band in the gel. ( C ) CC3 was expressed in cervical cancer cell lines; expression was higher in SiHa cells than in HeLa and CaSki cells.

Techniques Used: Polymerase Chain Reaction, Expressing

9) Product Images from "Immunologic Function and Molecular Insight of Recombinant Interleukin-18"

Article Title: Immunologic Function and Molecular Insight of Recombinant Interleukin-18

Journal: PLoS ONE

doi: 10.1371/journal.pone.0160321

Construction of the expression plasmid pPICZα-IL18WT and its mutants. (A) Strategy and schematic presentation of steps involved in the construction of expression plasmid pPICZa-IL18WT. A mature human IL-18 sequence was inserted into the expression plasmid at the Eco RI and Xba I sites. (B) Diagram showing the site-directed mutagenesis method. The mutant-strand was amplified by PCR and a wild-type DNA template was digested by Dpn I. The resulting annealed double-stranded nicked DNA molecules were transformed into E . coli DH5α and the nicked DNA was repaired.
Figure Legend Snippet: Construction of the expression plasmid pPICZα-IL18WT and its mutants. (A) Strategy and schematic presentation of steps involved in the construction of expression plasmid pPICZa-IL18WT. A mature human IL-18 sequence was inserted into the expression plasmid at the Eco RI and Xba I sites. (B) Diagram showing the site-directed mutagenesis method. The mutant-strand was amplified by PCR and a wild-type DNA template was digested by Dpn I. The resulting annealed double-stranded nicked DNA molecules were transformed into E . coli DH5α and the nicked DNA was repaired.

Techniques Used: Expressing, Plasmid Preparation, Sequencing, Mutagenesis, Amplification, Polymerase Chain Reaction, Transformation Assay

10) Product Images from "Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli"

Article Title: Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

Journal: Molecular Biology International

doi: 10.1155/2016/4732791

Restriction digestion of three different PA constructs. M, DNA marker; lane 1, PA-pQE30 digested with restriction enzymes Bam HI/ Kpn I; lane 2, PA-pPROEXHTa digested with restriction enzymes Bam HI/ Xho I; lane 3, PA-pET32c digested with restriction enzymes Bam HI/ Sal I.
Figure Legend Snippet: Restriction digestion of three different PA constructs. M, DNA marker; lane 1, PA-pQE30 digested with restriction enzymes Bam HI/ Kpn I; lane 2, PA-pPROEXHTa digested with restriction enzymes Bam HI/ Xho I; lane 3, PA-pET32c digested with restriction enzymes Bam HI/ Sal I.

Techniques Used: Construct, Marker

11) Product Images from "HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species"

Article Title: HybriFree: a robust and rapid method for the development of monoclonal antibodies from different host species

Journal: BMC Biotechnology

doi: 10.1186/s12896-016-0232-6

Construction and screening of intact IgG molecules. a The pQMCF IgG vector was constructed using single-step CPEC joining of 4 fragments: VH, VL, promoters/leaders and vector. The antibody heavy and light chains are expressed from the resulting vector as separate proteins that assemble naturally into IgG molecules secreted from mammalian cells. b . Western blot analysis of rabbit IgG secretion from CHOEBNALT85 cells transfected with pQMCF IgG library pool DNA constructed from VH and VL regions from a rabbit immunized with mouse CD48 protein. Goat polyclonal antibody against rabbit IgG heavy chain was used for the detection of free heavy chain in reduced sample conditions (DTT+) and of the assembled IgG molecule in non-reduced (DTT-) sample. c Mouse CD48 ELISA results obtained using serial dilutions of the same sample of library pool transfection media as primary antibody. d Distribution of positive and negative clones obtained from the screening of the library pool showed in the panel c . Three positive and two negative clones selected for sequencing are indicated. e Alignment of VL domain amino acid sequences of the positive and negative clones. CDR regions are underlined
Figure Legend Snippet: Construction and screening of intact IgG molecules. a The pQMCF IgG vector was constructed using single-step CPEC joining of 4 fragments: VH, VL, promoters/leaders and vector. The antibody heavy and light chains are expressed from the resulting vector as separate proteins that assemble naturally into IgG molecules secreted from mammalian cells. b . Western blot analysis of rabbit IgG secretion from CHOEBNALT85 cells transfected with pQMCF IgG library pool DNA constructed from VH and VL regions from a rabbit immunized with mouse CD48 protein. Goat polyclonal antibody against rabbit IgG heavy chain was used for the detection of free heavy chain in reduced sample conditions (DTT+) and of the assembled IgG molecule in non-reduced (DTT-) sample. c Mouse CD48 ELISA results obtained using serial dilutions of the same sample of library pool transfection media as primary antibody. d Distribution of positive and negative clones obtained from the screening of the library pool showed in the panel c . Three positive and two negative clones selected for sequencing are indicated. e Alignment of VL domain amino acid sequences of the positive and negative clones. CDR regions are underlined

Techniques Used: Plasmid Preparation, Construct, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, Clone Assay, Sequencing

12) Product Images from "Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes"

Article Title: Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0067952

URT pulse-chase assays with model N-end rule substrates in E. coli and V. vulnificus . The set of URT-based 3f DHFR-Ub R48 -X-βgal 3f fusions (X = Val, Leu, Arg, Asp) was assayed for the in vivo degradation of the released (by the yeast Ubp1 DUB) X-βgal proteins in E. coli ( A, B ) and in V. vulnificus ( C, D ) using 35 S-pulse-chases ( A, C ) and their quantification ( B, D ), as described in Materials and Methods. The bands of the 110 kDa X-βgal test proteins and the 33 kDa 3f DHFR-Ub R48 reference protein are indicated on the left. Designations in B and D : squares, Val-βgal; rhombs, Leu-βgal; triangles, Arg-βgal; crosses, Asp-βgal. E . pKP55-X, encoding the S. cerevisiae Ubp1 DUB and 3f DHFR-Ub R48 -X-βgal 3f URT-based fusions. Other notations on the map denote specific bacterial genes. The nucleotide sequences of pKP77 and pKP55-X are available in GenBank (JX181779 and JX181780). In addition, Table S3 contains the nucleotide sequence of pKP55-X.
Figure Legend Snippet: URT pulse-chase assays with model N-end rule substrates in E. coli and V. vulnificus . The set of URT-based 3f DHFR-Ub R48 -X-βgal 3f fusions (X = Val, Leu, Arg, Asp) was assayed for the in vivo degradation of the released (by the yeast Ubp1 DUB) X-βgal proteins in E. coli ( A, B ) and in V. vulnificus ( C, D ) using 35 S-pulse-chases ( A, C ) and their quantification ( B, D ), as described in Materials and Methods. The bands of the 110 kDa X-βgal test proteins and the 33 kDa 3f DHFR-Ub R48 reference protein are indicated on the left. Designations in B and D : squares, Val-βgal; rhombs, Leu-βgal; triangles, Arg-βgal; crosses, Asp-βgal. E . pKP55-X, encoding the S. cerevisiae Ubp1 DUB and 3f DHFR-Ub R48 -X-βgal 3f URT-based fusions. Other notations on the map denote specific bacterial genes. The nucleotide sequences of pKP77 and pKP55-X are available in GenBank (JX181779 and JX181780). In addition, Table S3 contains the nucleotide sequence of pKP55-X.

Techniques Used: Pulse Chase, In Vivo, Sequencing

13) Product Images from "β-nicotinamide mononucleotide (NMN) production in Escherichia coli"

Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

Journal: Scientific Reports

doi: 10.1038/s41598-018-30792-0

pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.
Figure Legend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

Techniques Used: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay

14) Product Images from "GRAS Proteins Form a DNA Binding Complex to Induce Gene Expression during Nodulation Signaling in Medicago truncatula [W]"

Article Title: GRAS Proteins Form a DNA Binding Complex to Induce Gene Expression during Nodulation Signaling in Medicago truncatula [W]

Journal: The Plant Cell

doi: 10.1105/tpc.108.064501

NSP1 and NSP2 Form Heteropolymers in Planta. (A) to (C) N. benthamiana leaves were cotransformed with YFP N -NSP1 and YFP C -NSP2 (A) , YFP N -NSP1 and YFP C -NSP2-LHRI (B) , and YFP N -NSP1 and YFP C -NSP2ΔLHRI (C) . Overlays of confocal YFP and bright-field images of epidermal N. benthamiana leaf cells are shown. (A) and (B) show the nuclear localization of the NSP1 and NSP2 interaction. Removal of the LHRI domain of NSP2 abolished the interaction with NSP1 (C) . The inset of (C) confirms protein stability of (a) YFP C -NSP2 (65 kD) and (b) YFP C -NSP2ΔLHRI (58 kD) in these N. benthamiana transient expression studies. Bars = 40 μm. (D) NSP1-3xHA alone and NSP2-FLAG with NSP1-3xHA were transiently expressed in N. benthamiana leaves. Leaves were harvested 3 d after infiltration, and equal amounts of total protein extracts (input) were subjected to immunoprecipitation with α-FLAG antibodies. Analysis of the elution revealed coimmunoprecipitation of NSP1-3xHA with NSP2-FLAG that was not observed in the leaves transformed with NSP1-3xHA alone.
Figure Legend Snippet: NSP1 and NSP2 Form Heteropolymers in Planta. (A) to (C) N. benthamiana leaves were cotransformed with YFP N -NSP1 and YFP C -NSP2 (A) , YFP N -NSP1 and YFP C -NSP2-LHRI (B) , and YFP N -NSP1 and YFP C -NSP2ΔLHRI (C) . Overlays of confocal YFP and bright-field images of epidermal N. benthamiana leaf cells are shown. (A) and (B) show the nuclear localization of the NSP1 and NSP2 interaction. Removal of the LHRI domain of NSP2 abolished the interaction with NSP1 (C) . The inset of (C) confirms protein stability of (a) YFP C -NSP2 (65 kD) and (b) YFP C -NSP2ΔLHRI (58 kD) in these N. benthamiana transient expression studies. Bars = 40 μm. (D) NSP1-3xHA alone and NSP2-FLAG with NSP1-3xHA were transiently expressed in N. benthamiana leaves. Leaves were harvested 3 d after infiltration, and equal amounts of total protein extracts (input) were subjected to immunoprecipitation with α-FLAG antibodies. Analysis of the elution revealed coimmunoprecipitation of NSP1-3xHA with NSP2-FLAG that was not observed in the leaves transformed with NSP1-3xHA alone.

Techniques Used: Expressing, Immunoprecipitation, Transformation Assay

NSP1 and NSP2 Associate with the ENOD11 Promoter in Vivo . ChIP followed by PCR show in vivo association of both NSP1 and NSP2 with the ENOD11 promoter. NSP1 and NSP2 were purified from M. truncatula root extracts using their native antibodies; c-Myc antibodies were used as a negative control. Pretreatment with Nod factor enhanced the binding of NSP1 to the ENOD11 promoter and revealed an NSP2 association that we propose is via the interaction with NSP1. An equivalent analysis in nsp1-1 and nsp2-2 mutants revealed no association of NSP1 or NSP2 with the ENOD11 promoter.
Figure Legend Snippet: NSP1 and NSP2 Associate with the ENOD11 Promoter in Vivo . ChIP followed by PCR show in vivo association of both NSP1 and NSP2 with the ENOD11 promoter. NSP1 and NSP2 were purified from M. truncatula root extracts using their native antibodies; c-Myc antibodies were used as a negative control. Pretreatment with Nod factor enhanced the binding of NSP1 to the ENOD11 promoter and revealed an NSP2 association that we propose is via the interaction with NSP1. An equivalent analysis in nsp1-1 and nsp2-2 mutants revealed no association of NSP1 or NSP2 with the ENOD11 promoter.

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Purification, Negative Control, Binding Assay

NSP1 Associates with ENOD Promoters in Vitro . (A) to (C) EMSA with NSP1 and NSP2 proteins using radiolabeled ENOD promoter probes. The promoter regions of ENOD11 (−1046 to +3) (A) , NIN (−892 to −13) (B) , and ERN1 (−862 to −29) (C) were used. The free ENOD promoter probe is present at the bottom of each image. The band shift, indicated with an arrow, revealed NSP1 association with the promoter of ENOD11 (A) , NIN (B) , and ERN1 (C) that is absent in the GST control and in NSP2. In (A) , unlabeled ENOD11 fragment was used as competitor DNA. c10x and c50x, unlabeled competitor DNA in 10- and 50-fold excess, respectively. (D) RT-PCR showing that the S. meliloti induction of NIN and ERN1 is absent in nsp1-1 and nsp2-2 mutants. Actin was amplified as a control. dpi, days post inoculation.
Figure Legend Snippet: NSP1 Associates with ENOD Promoters in Vitro . (A) to (C) EMSA with NSP1 and NSP2 proteins using radiolabeled ENOD promoter probes. The promoter regions of ENOD11 (−1046 to +3) (A) , NIN (−892 to −13) (B) , and ERN1 (−862 to −29) (C) were used. The free ENOD promoter probe is present at the bottom of each image. The band shift, indicated with an arrow, revealed NSP1 association with the promoter of ENOD11 (A) , NIN (B) , and ERN1 (C) that is absent in the GST control and in NSP2. In (A) , unlabeled ENOD11 fragment was used as competitor DNA. c10x and c50x, unlabeled competitor DNA in 10- and 50-fold excess, respectively. (D) RT-PCR showing that the S. meliloti induction of NIN and ERN1 is absent in nsp1-1 and nsp2-2 mutants. Actin was amplified as a control. dpi, days post inoculation.

Techniques Used: In Vitro, Electrophoretic Mobility Shift Assay, Reverse Transcription Polymerase Chain Reaction, Amplification

NSP1 and NSP2 Form Homopolymers. (A) NSP1-3xHA alone or NSP1-FLAG and NSP1-3xHA were transiently expressed under the control of the 35S promoter in N. benthamiana leaves. Leaves were harvested 3 d after infiltration and NSP1-FLAG immunoprecipitated (elution) from total protein extracts (input). Coimmunoprecipitation of NSP1-3xHA indicated an interaction of NSP1 with itself. (B) In a similar manner, N. benthamiana leaves were transformed with NSP2-3xHA or NSP2∷GFP alone, NSP2∷3xHA and NSP2-FLAG , or NSP2-GFP and NSP2∷FLAG . NSP2-FLAG was immunoprecipitated (elution) from total protein extracts (input) 3 d after infiltration. Coimmunoprecipitation of NSP2-GFP and NSP2-3xHA with NSP2-FLAG revealed homopolymerization of this protein. The input shown is from the NSP2-FLAG/NSP2-3xHA experiment. Similar inputs were observed with the NSP2-FLAG/NSP2-GFP experiment. (C) Leaves of N. benthamiana were transformed with YFP N -NSP1 and YFP C -NSP1 . Yellow fluorescence in the nuclei indicates homopolymerization of NSP1. Bar = 40 μm.
Figure Legend Snippet: NSP1 and NSP2 Form Homopolymers. (A) NSP1-3xHA alone or NSP1-FLAG and NSP1-3xHA were transiently expressed under the control of the 35S promoter in N. benthamiana leaves. Leaves were harvested 3 d after infiltration and NSP1-FLAG immunoprecipitated (elution) from total protein extracts (input). Coimmunoprecipitation of NSP1-3xHA indicated an interaction of NSP1 with itself. (B) In a similar manner, N. benthamiana leaves were transformed with NSP2-3xHA or NSP2∷GFP alone, NSP2∷3xHA and NSP2-FLAG , or NSP2-GFP and NSP2∷FLAG . NSP2-FLAG was immunoprecipitated (elution) from total protein extracts (input) 3 d after infiltration. Coimmunoprecipitation of NSP2-GFP and NSP2-3xHA with NSP2-FLAG revealed homopolymerization of this protein. The input shown is from the NSP2-FLAG/NSP2-3xHA experiment. Similar inputs were observed with the NSP2-FLAG/NSP2-GFP experiment. (C) Leaves of N. benthamiana were transformed with YFP N -NSP1 and YFP C -NSP1 . Yellow fluorescence in the nuclei indicates homopolymerization of NSP1. Bar = 40 μm.

Techniques Used: Immunoprecipitation, Transformation Assay, Fluorescence

15) Product Images from "Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase"

Article Title: Cloning, Expression and Purification of Pseudomonas putida ATCC12633 Creatinase

Journal: Avicenna Journal of Medical Biotechnology

doi:

Lane 1: PCR product of Cre gene, lane 2: pET28a-cre plasmid extraction result, lane 3: DNA ladder, lane 4: double digestion of recombinant pET28a-cre by NheI and XhoI. Products were electrophoresed on 0.7% agarose gel.
Figure Legend Snippet: Lane 1: PCR product of Cre gene, lane 2: pET28a-cre plasmid extraction result, lane 3: DNA ladder, lane 4: double digestion of recombinant pET28a-cre by NheI and XhoI. Products were electrophoresed on 0.7% agarose gel.

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Recombinant, Agarose Gel Electrophoresis

16) Product Images from "Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu–Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice"

Article Title: Immunogenicity of a Multi-Epitope DNA Vaccine Encoding Epitopes from Cu–Zn Superoxide Dismutase and Open Reading Frames of Brucella abortus in Mice

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2017.00125

MEB DNA vaccine design and identification of rMEB protein . (A) Multi-epitope vaccine sequence spaced by GDGDG linker sequence. (B) SDS-PAGE analysis of rMEB. Lane 1, marker; lane 2, total proteins obtained from insoluble extract from Escherichia coli transformed with pQE80L-MEBe plasmid; lane 3, eluent from Ni 2+ -chelated affinity chromatography of the insoluble extract from E. coli ; lane 4, total proteins obtained from the soluble extract from E. coli transformed with pQE80L-MEBe plasmid; lane 5, eluent from Ni 2+ -chelated affinity chromatography of the soluble extract from E. coli . (C) Western blot analysis of rMEB with anti-His-tag monoclonal antibody.
Figure Legend Snippet: MEB DNA vaccine design and identification of rMEB protein . (A) Multi-epitope vaccine sequence spaced by GDGDG linker sequence. (B) SDS-PAGE analysis of rMEB. Lane 1, marker; lane 2, total proteins obtained from insoluble extract from Escherichia coli transformed with pQE80L-MEBe plasmid; lane 3, eluent from Ni 2+ -chelated affinity chromatography of the insoluble extract from E. coli ; lane 4, total proteins obtained from the soluble extract from E. coli transformed with pQE80L-MEBe plasmid; lane 5, eluent from Ni 2+ -chelated affinity chromatography of the soluble extract from E. coli . (C) Western blot analysis of rMEB with anti-His-tag monoclonal antibody.

Techniques Used: Sequencing, SDS Page, Marker, Transformation Assay, Plasmid Preparation, Affinity Chromatography, Western Blot

17) Product Images from "Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis"

Article Title: Overexpression and Suppression of Artemisia annua 4-Hydroxy-3-Methylbut-2-enyl Diphosphate Reductase 1 Gene (AaHDR1) Differentially Regulate Artemisinin and Terpenoid Biosynthesis

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.00077

Expression pattern of AaHDR1 and subcellular localization. (A) Semi-quantitative RT-PCR (upper panel) and real time quantitative RT-PCR (bottom panel) analysis of AaHDR1 expression levels in different organs of A. annua , OF: full open flowers, FB: flower buds, L2: young leaves from plants grown on soil in the phytotron, L1, S and R: stems and roots of 8-week old seedlings grown on MS-medium contained in baby jars, Con: buffer control without the first strand of cDNA, a–f: different low case labeling indicating significant difference ( p -value
Figure Legend Snippet: Expression pattern of AaHDR1 and subcellular localization. (A) Semi-quantitative RT-PCR (upper panel) and real time quantitative RT-PCR (bottom panel) analysis of AaHDR1 expression levels in different organs of A. annua , OF: full open flowers, FB: flower buds, L2: young leaves from plants grown on soil in the phytotron, L1, S and R: stems and roots of 8-week old seedlings grown on MS-medium contained in baby jars, Con: buffer control without the first strand of cDNA, a–f: different low case labeling indicating significant difference ( p -value

Techniques Used: Expressing, Quantitative RT-PCR, Mass Spectrometry, Labeling

18) Product Images from "Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice"

Article Title: Rv2629 Overexpression Delays Mycobacterium smegmatis and Mycobacteria tuberculosis Entry into Log-Phase and Increases Pathogenicity of Mycobacterium smegmatis in Mice

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02231

The expression of Rv2629 modulates the growth of M. smegmatis under aerobic conditions. The growth curve of recombinant M. smegmatis strains that exhibited reduced expression of Rv2629 and overexpression of the Rv2629 W and/or the Rv2629 M genes, respectively. The growth of the strains was measured by harvesting cultures at 3-h intervals and subsequent measurement of the absorbance values at 600 nm (OD 600 ). The abbreviations were: MS (wild type), MSP (control strain transformed with the pMV261 plasmid), and MSpACT (control strain transformed with the pACT plasmid). The results indicated (A) comparatively slow growth of MSW . (B) Comparatively rapid growth of MSL . The data are representative of two independent experiments with two samples in each experiment which shows similar results.
Figure Legend Snippet: The expression of Rv2629 modulates the growth of M. smegmatis under aerobic conditions. The growth curve of recombinant M. smegmatis strains that exhibited reduced expression of Rv2629 and overexpression of the Rv2629 W and/or the Rv2629 M genes, respectively. The growth of the strains was measured by harvesting cultures at 3-h intervals and subsequent measurement of the absorbance values at 600 nm (OD 600 ). The abbreviations were: MS (wild type), MSP (control strain transformed with the pMV261 plasmid), and MSpACT (control strain transformed with the pACT plasmid). The results indicated (A) comparatively slow growth of MSW . (B) Comparatively rapid growth of MSL . The data are representative of two independent experiments with two samples in each experiment which shows similar results.

Techniques Used: Expressing, Recombinant, Over Expression, Mass Spectrometry, Transformation Assay, Plasmid Preparation

19) Product Images from "High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy"

Article Title: High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

Journal:

doi: 10.1016/j.bbamem.2007.09.009

13 C- 13 C spectrum of CYP98A3 with 10 ms DARR mixing
Figure Legend Snippet: 13 C- 13 C spectrum of CYP98A3 with 10 ms DARR mixing

Techniques Used: Mass Spectrometry

SSNMR 1D spectra of CYP98A3
Figure Legend Snippet: SSNMR 1D spectra of CYP98A3

Techniques Used:

13 C- 13 C INADEQUATE spectrum of CYP98A3
Figure Legend Snippet: 13 C- 13 C INADEQUATE spectrum of CYP98A3

Techniques Used:

SDS-PAGE gel electrophoresis and CO-difference analysis of 13 C, 15 N-labeled His 4 -CYP98A3
Figure Legend Snippet: SDS-PAGE gel electrophoresis and CO-difference analysis of 13 C, 15 N-labeled His 4 -CYP98A3

Techniques Used: SDS Page, Nucleic Acid Electrophoresis, Labeling

20) Product Images from "Stationary-Phase Quorum-Sensing Signals Affect Autoinducer-2 and Gene Expression in Escherichia coli"

Article Title: Stationary-Phase Quorum-Sensing Signals Affect Autoinducer-2 and Gene Expression in Escherichia coli

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.70.4.2038-2043.2004

Comparison of gene expression profiles affected by FCM and ACM.
Figure Legend Snippet: Comparison of gene expression profiles affected by FCM and ACM.

Techniques Used: Expressing

Comparison of gene expression profiles affected by FCM and ACM.
Figure Legend Snippet: Comparison of gene expression profiles affected by FCM and ACM.

Techniques Used: Expressing

Comparison of gene expression profiles affected by FCM and ACM.
Figure Legend Snippet: Comparison of gene expression profiles affected by FCM and ACM.

Techniques Used: Expressing

Comparison of gene expression profiles affected by FCM and ACM.
Figure Legend Snippet: Comparison of gene expression profiles affected by FCM and ACM.

Techniques Used: Expressing

21) Product Images from "Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly"

Article Title: Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly

Journal: Biochemistry

doi: 10.1021/bi101005r

Sucrose density gradient profiles and in vitro time course KsgA methylation assay of 30S subunit substrates. (A) Sucrose gradient profile of 30S subunits isolated from MRE600 E. coli strain made kasugamycin resistant (30S UnTag,UM ) (B) Sucrose gradient
Figure Legend Snippet: Sucrose density gradient profiles and in vitro time course KsgA methylation assay of 30S subunit substrates. (A) Sucrose gradient profile of 30S subunits isolated from MRE600 E. coli strain made kasugamycin resistant (30S UnTag,UM ) (B) Sucrose gradient

Techniques Used: In Vitro, Methylation, Isolation

22) Product Images from "PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa"

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00548

Role of PruR in P. aeruginosa infection in a mouse acute pneumonia model and bacterial resistance to oxidative stress. (A) Mice were inoculated intranasally with 4 × 10 7 CFU bacteria of indicated strains. The mice were monitored for 5 days after the infection. The data were from 13 mice per strain. * p
Figure Legend Snippet: Role of PruR in P. aeruginosa infection in a mouse acute pneumonia model and bacterial resistance to oxidative stress. (A) Mice were inoculated intranasally with 4 × 10 7 CFU bacteria of indicated strains. The mice were monitored for 5 days after the infection. The data were from 13 mice per strain. * p

Techniques Used: Infection, Mouse Assay

Role of PutA in P. aeruginosa infection in a mouse acute pneumonia model. Mice were inoculated intranasally with 4 × 10 7 CFU bacteria of indicated strains. The mice were monitored for 5 days after the infection. The data were from 13 mice per strain. * p
Figure Legend Snippet: Role of PutA in P. aeruginosa infection in a mouse acute pneumonia model. Mice were inoculated intranasally with 4 × 10 7 CFU bacteria of indicated strains. The mice were monitored for 5 days after the infection. The data were from 13 mice per strain. * p

Techniques Used: Infection, Mouse Assay

Schematic diagram of putA gene in P. aeruginosa . Genes are represented by open arrows. The gene designations are above the arrows. The length of each gene is indicated below the arrow. The length of each intergenic region is indicated below the bar and underlined. Data were from P. aeruginosa reference strain PAO1 (Winsor et al., 2016 ). The length of each gene and intergenic region is conserved between the PAO1 strain and P. aeruginosa PAK strain. The homology of the fragment (from the intergenic region of asrA and pruR gene to the putP gene, 8,605 bp) between the PAO1 strain and the PAK strain is 99.78%.
Figure Legend Snippet: Schematic diagram of putA gene in P. aeruginosa . Genes are represented by open arrows. The gene designations are above the arrows. The length of each gene is indicated below the arrow. The length of each intergenic region is indicated below the bar and underlined. Data were from P. aeruginosa reference strain PAO1 (Winsor et al., 2016 ). The length of each gene and intergenic region is conserved between the PAO1 strain and P. aeruginosa PAK strain. The homology of the fragment (from the intergenic region of asrA and pruR gene to the putP gene, 8,605 bp) between the PAO1 strain and the PAK strain is 99.78%.

Techniques Used:

23) Product Images from "Genetic manipulation of Staphylococcus aureus"

Article Title: Genetic manipulation of Staphylococcus aureus

Journal: Current protocols in microbiology

doi: 10.1002/9780471729259.mc09c03s32

Map of transposon bursa aurealis and plasmids used for delivery along with summary of steps involved in the mapping of insertion sites by inverse PCR. (A) Bursa aurealis (3.2 kbp), a mini-mariner transposable element, was cloned into pTS2, with a temperature-sensitive plasmid replicon (rep ts ) and chloramphenicol resistance gene cat to generate pBursa (7,383 bp). Bursa aurealis encompasses mariner terminal inverted repeats (TIR), R6K replication origin (oriV) for replication in E. coli , and erythromycin-resistance determinant ermC , an rRNA methylase that allows selection in both E. coli and S. aureus . The position of the most terminal site for the restriction enzyme Aci I is indicated as well as the site of hybridization and nucleotide sequence of primer Martn-F (F). Plasmid pFA545 (10,079 bp) encodes the mariner transposase tnp and is a derivative of pSPT181, a shuttle vector consisting of pSP64 with ampicillin resistance ( bla ) for replication and selection in E. coli , and pRN8103, a temperature-sensitive derivative of pT181 (rep ts ) and tetracycline-resistance marker ( tetB tetD ). The presence of rep ts and tetBD allows for replication of pFA545 in S. aureus and other Gram-positive bacteria. (B) Mapping insertion sites by inverse PCR. Genome DNA from a candidate mutant strain is isolated and digested with Aci I. Next, fragment self-ligation and inverse PCR are performed using DNA ligase and primers Martn-F (F) and Martn-ermR (R). PCR products are subjected to DNA sequence analysis using primer Martn-F (F).
Figure Legend Snippet: Map of transposon bursa aurealis and plasmids used for delivery along with summary of steps involved in the mapping of insertion sites by inverse PCR. (A) Bursa aurealis (3.2 kbp), a mini-mariner transposable element, was cloned into pTS2, with a temperature-sensitive plasmid replicon (rep ts ) and chloramphenicol resistance gene cat to generate pBursa (7,383 bp). Bursa aurealis encompasses mariner terminal inverted repeats (TIR), R6K replication origin (oriV) for replication in E. coli , and erythromycin-resistance determinant ermC , an rRNA methylase that allows selection in both E. coli and S. aureus . The position of the most terminal site for the restriction enzyme Aci I is indicated as well as the site of hybridization and nucleotide sequence of primer Martn-F (F). Plasmid pFA545 (10,079 bp) encodes the mariner transposase tnp and is a derivative of pSPT181, a shuttle vector consisting of pSP64 with ampicillin resistance ( bla ) for replication and selection in E. coli , and pRN8103, a temperature-sensitive derivative of pT181 (rep ts ) and tetracycline-resistance marker ( tetB tetD ). The presence of rep ts and tetBD allows for replication of pFA545 in S. aureus and other Gram-positive bacteria. (B) Mapping insertion sites by inverse PCR. Genome DNA from a candidate mutant strain is isolated and digested with Aci I. Next, fragment self-ligation and inverse PCR are performed using DNA ligase and primers Martn-F (F) and Martn-ermR (R). PCR products are subjected to DNA sequence analysis using primer Martn-F (F).

Techniques Used: Inverse PCR, Clone Assay, Plasmid Preparation, Selection, Hybridization, Sequencing, Marker, Mutagenesis, Isolation, Ligation, Polymerase Chain Reaction

24) Product Images from "PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis"

Article Title: PpiA, a Surface PPIase of the Cyclophilin Family in Lactococcus lactis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0033516

Protein sequence alignment between lactococcal PpiA protein and related cyclophilins. The sequences of some cyclophilins: Homo sapiens hCyp18 (Accession n° P62937), E. coli PpiA (P0AFL3), S. pneumoniae SlrA (NP_358273), and L. lactis PpiA (from strain IL1403: NP_266521.1 or strain MG1363: YP_001031737.1), are shown. Sequence alignment was performed using MultiAlin and manually improved to align the N-terminal hydrophobic domains of the three bacterial exported PPIases, and to take into account a previously published alignment [12] . Identical amino acids are marked with asterisks, and gaps with dash characters. The amino acids of the catalytic center of hCyp18 are marked in bold (in hCyp18 and all the proteins where they are conserved). The insertion sequence that is specific for S. pneumoniae SlrA compared to E. coli PpiA and hCyp18 [12] , and conserved in L. lactis PpiA, is boxed. The N-terminal hydrophobic sequence of lactococcal PpiA proteins is double underlined. PpiA peptides released by shaving treatment of MG1363 cells are underlined.
Figure Legend Snippet: Protein sequence alignment between lactococcal PpiA protein and related cyclophilins. The sequences of some cyclophilins: Homo sapiens hCyp18 (Accession n° P62937), E. coli PpiA (P0AFL3), S. pneumoniae SlrA (NP_358273), and L. lactis PpiA (from strain IL1403: NP_266521.1 or strain MG1363: YP_001031737.1), are shown. Sequence alignment was performed using MultiAlin and manually improved to align the N-terminal hydrophobic domains of the three bacterial exported PPIases, and to take into account a previously published alignment [12] . Identical amino acids are marked with asterisks, and gaps with dash characters. The amino acids of the catalytic center of hCyp18 are marked in bold (in hCyp18 and all the proteins where they are conserved). The insertion sequence that is specific for S. pneumoniae SlrA compared to E. coli PpiA and hCyp18 [12] , and conserved in L. lactis PpiA, is boxed. The N-terminal hydrophobic sequence of lactococcal PpiA proteins is double underlined. PpiA peptides released by shaving treatment of MG1363 cells are underlined.

Techniques Used: Sequencing

25) Product Images from "Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs"

Article Title: Absolute quantification of the budding yeast transcriptome by means of competitive PCR between genomic and complementary DNAs

Journal: BMC Genomics

doi: 10.1186/1471-2164-9-574

Calibration of GATC-PCR between genomic DNA and cDNA . (A) Competitive amplification of GCN4 between genomic DNA and cDNA. (B) Standard RNAs used for competitive PCR determination of mRNA copy number. (C) Comparison of absolute amounts of eight mRNAs determined by real-time PCR and GATC-PCR. For real-time PCR, we used each GSP for the first strand cDNA synthesis. The GATC-PCR data were calibrated by the competitive PCR quantification of GCN4 mRNA using the standard RNA set (Figure 2B, Table 3 ).
Figure Legend Snippet: Calibration of GATC-PCR between genomic DNA and cDNA . (A) Competitive amplification of GCN4 between genomic DNA and cDNA. (B) Standard RNAs used for competitive PCR determination of mRNA copy number. (C) Comparison of absolute amounts of eight mRNAs determined by real-time PCR and GATC-PCR. For real-time PCR, we used each GSP for the first strand cDNA synthesis. The GATC-PCR data were calibrated by the competitive PCR quantification of GCN4 mRNA using the standard RNA set (Figure 2B, Table 3 ).

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

Generalized Adaptor-Tagged Competitive PCR (GATC-PCR) . (A) Gene-specific primer (GSP)-dependent amplification from Y-shaped adaptor-tagged template. (B) An example of GATC-PCR. Genomic DNA and cDNA digested with Mbo I were ligated with adaptor A/C and B/C (Table 2 ), respectively, and used for GATC-PCR. The products of four assays (blue, green, red, and black) and a size standard (orange) were separated on ABI 3730 Genetic Analyzer. The fast- and slow-migrating peaks of each pair correspond to the signals from genomic DNA and cDNA, respectively. (C) Linearity of GATC-PCR from genomic DNA templates. Genomic DNAs extracted from the wild and gcn4 Δ cells were combined at appropriate ratios to prepare a series of genomic DNAs containing 0, 0.25, 0.5, 0.75, and 1 copy of GCN4 per haploid on average, digested with Mbo I, and ligated to the adaptors A/C and B/C (Table 2 ). Various combinations of the A/C- and B/C-tagged templates were mixed in a 1:1 ratio, while keeping the total amount equivalent to 3,000 haploid cells, and subjected to GATC-PCR using a GCN4 -specific primer. (D) Linearity of GATC-PCR from cDNA templates. An experiment similar to the one shown in (C) was conducted using cDNAs, instead of genomic DNA, prepared from the wild and gcn4 Δ cells.
Figure Legend Snippet: Generalized Adaptor-Tagged Competitive PCR (GATC-PCR) . (A) Gene-specific primer (GSP)-dependent amplification from Y-shaped adaptor-tagged template. (B) An example of GATC-PCR. Genomic DNA and cDNA digested with Mbo I were ligated with adaptor A/C and B/C (Table 2 ), respectively, and used for GATC-PCR. The products of four assays (blue, green, red, and black) and a size standard (orange) were separated on ABI 3730 Genetic Analyzer. The fast- and slow-migrating peaks of each pair correspond to the signals from genomic DNA and cDNA, respectively. (C) Linearity of GATC-PCR from genomic DNA templates. Genomic DNAs extracted from the wild and gcn4 Δ cells were combined at appropriate ratios to prepare a series of genomic DNAs containing 0, 0.25, 0.5, 0.75, and 1 copy of GCN4 per haploid on average, digested with Mbo I, and ligated to the adaptors A/C and B/C (Table 2 ). Various combinations of the A/C- and B/C-tagged templates were mixed in a 1:1 ratio, while keeping the total amount equivalent to 3,000 haploid cells, and subjected to GATC-PCR using a GCN4 -specific primer. (D) Linearity of GATC-PCR from cDNA templates. An experiment similar to the one shown in (C) was conducted using cDNAs, instead of genomic DNA, prepared from the wild and gcn4 Δ cells.

Techniques Used: Polymerase Chain Reaction, Amplification

26) Product Images from "Pseudomonas aeruginosa Elastase Provides an Escape from Phagocytosis by Degrading the Pulmonary Surfactant Protein-A"

Article Title: Pseudomonas aeruginosa Elastase Provides an Escape from Phagocytosis by Degrading the Pulmonary Surfactant Protein-A

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027091

Δ lasB mutant bacteria are resistant to SP-A-mediated membrane permeabilization. Membrane permeabilization assays were performed with 1×10 8 of E. coli DH5α or P. aeruginosa exposed to hSP-A (50 µg/ml) for 120 min. Three independent experiments were performed in triplicates. The mean + standard deviation from one representative experiment is shown. The membrane permeabilization activity of hSP-A against PAO1, Δ lasB and PDO240lasB was not statistically different among all three P. aeruginosa strains. * p
Figure Legend Snippet: Δ lasB mutant bacteria are resistant to SP-A-mediated membrane permeabilization. Membrane permeabilization assays were performed with 1×10 8 of E. coli DH5α or P. aeruginosa exposed to hSP-A (50 µg/ml) for 120 min. Three independent experiments were performed in triplicates. The mean + standard deviation from one representative experiment is shown. The membrane permeabilization activity of hSP-A against PAO1, Δ lasB and PDO240lasB was not statistically different among all three P. aeruginosa strains. * p

Techniques Used: Mutagenesis, Standard Deviation, Activity Assay

27) Product Images from "Clostridium pasteurianum F1Fo ATP Synthase: Operon, Composition, and Some Properties"

Article Title: Clostridium pasteurianum F1Fo ATP Synthase: Operon, Composition, and Some Properties

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.18.5527-5535.2003

Immunoblots of soluble F 1 F o (A) and F 1 (B) from C . pasteurianum (Cp) and E . coli DH5α (Ec) after reactions with antibodies against purified F 1Mt . (A) Soluble F 1 F oCp after precipitation with ammonium sulfate from DM extracts (1.0% [wt/vol]) of C . pasteurianum membranes (30 μg) and soluble F 1 F oEc in DM extracts (1.0% [wt/vol]) of E . coli membranes (30 μg). (B) Partially purified F 1Cp (30 μg) and F 1Ec (30 μg). Proteins were separated by native PAGE (7.5% acrylamide) and transblotted onto polyvinylidene difluoride membranes prior to immunoreactions.
Figure Legend Snippet: Immunoblots of soluble F 1 F o (A) and F 1 (B) from C . pasteurianum (Cp) and E . coli DH5α (Ec) after reactions with antibodies against purified F 1Mt . (A) Soluble F 1 F oCp after precipitation with ammonium sulfate from DM extracts (1.0% [wt/vol]) of C . pasteurianum membranes (30 μg) and soluble F 1 F oEc in DM extracts (1.0% [wt/vol]) of E . coli membranes (30 μg). (B) Partially purified F 1Cp (30 μg) and F 1Ec (30 μg). Proteins were separated by native PAGE (7.5% acrylamide) and transblotted onto polyvinylidene difluoride membranes prior to immunoreactions.

Techniques Used: Western Blot, Purification, Clear Native PAGE

28) Product Images from "Cloning and DNA Sequence Analysis of an Immunogenic Glucose-Galactose MglB Lipoprotein Homologue from Brachyspira pilosicoli, the Agent of Colonic Spirochetosis †"

Article Title: Cloning and DNA Sequence Analysis of an Immunogenic Glucose-Galactose MglB Lipoprotein Homologue from Brachyspira pilosicoli, the Agent of Colonic Spirochetosis †

Journal: Infection and Immunity

doi:

Western blot (A) and autoradiograph (B) of lipoprotein-labeled recombinant B. pilosicoli MglB expressed in E. coli DH5α transformed with plasmid pPZD1003-36 or plasmid pCR2.1 as a negative control. (A) E. coli DH5α/pPZD1003-36 or E. coli DH5α/pCR2.1 control cells grown in LB medium with or without ampicillin (optical density at 600 nm of 0.5), respectively, were separated by SDS–10% PAGE, blotted onto nitrocellulose membranes, and reacted with B. pilosicoli hyperimmune swine serum preabsorbed with E. coli DH5α, followed by biotin-labeled goat anti-swine IgG, peroxidase-labeled streptavidin, and substrate. Lanes: 1, E. coli DH5α/pCR2.1; 2, E. coli DH5α/pPZD1003-36; 3, whole-cell lysate of B. pilosicoli strain SP16. (B) Same as panel A except that the cells were labeled for 3 h with [ 3 H]palmitate before separation by SDS–12.5% PAGE, drying, and autoradiography. Lanes: 1, E. coli DH5α/pCR2.1; 2, E. coli DH5α/pPZD1003-36. Molecular weights in thousands are indicated.
Figure Legend Snippet: Western blot (A) and autoradiograph (B) of lipoprotein-labeled recombinant B. pilosicoli MglB expressed in E. coli DH5α transformed with plasmid pPZD1003-36 or plasmid pCR2.1 as a negative control. (A) E. coli DH5α/pPZD1003-36 or E. coli DH5α/pCR2.1 control cells grown in LB medium with or without ampicillin (optical density at 600 nm of 0.5), respectively, were separated by SDS–10% PAGE, blotted onto nitrocellulose membranes, and reacted with B. pilosicoli hyperimmune swine serum preabsorbed with E. coli DH5α, followed by biotin-labeled goat anti-swine IgG, peroxidase-labeled streptavidin, and substrate. Lanes: 1, E. coli DH5α/pCR2.1; 2, E. coli DH5α/pPZD1003-36; 3, whole-cell lysate of B. pilosicoli strain SP16. (B) Same as panel A except that the cells were labeled for 3 h with [ 3 H]palmitate before separation by SDS–12.5% PAGE, drying, and autoradiography. Lanes: 1, E. coli DH5α/pCR2.1; 2, E. coli DH5α/pPZD1003-36. Molecular weights in thousands are indicated.

Techniques Used: Western Blot, Autoradiography, Labeling, Recombinant, Transformation Assay, Plasmid Preparation, Negative Control, Polyacrylamide Gel Electrophoresis

29) Product Images from "Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi"

Article Title: Subset of Hybrid Eukaryotic Proteins Is Exported by the Type I Secretion System of Erwinia chrysanthemi

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.4.1346-1358.2001

Expression of GFP- and endochitinase-181CTPB hybrids in E. coli . Protein hybrids were separated by SDS–12% PAGE; this was followed by Western blotting revealed with anti-protease B antibodies (diluted 1:1,000) and a second antibody (goat anti-rabbit immunoglobulin G coupled to peroxidase; diluted 1:1,000). Bacterial cultures harboring plasmids pGFP-181CTPB in E. coli HB101 cells and pCHIT-181CTPB in E. coli C600 were grown in LB medium with 100 μg of ampicillin/ml at 37°C until the OD 600 ). The gel was loaded with 25 μl. The same volume of supernatant (lanes S) was precipitated with 10% TCA, and the pellets were washed twice with acetone, dissolved, and loaded as described for lysates. Std, GIBCO-BRL Benchmark molecular size standard in panels A through C and GIBCO-BRL high-molecular-weight standard in panels D and E. (A) GFP-181CTPB secretion in E. coli HB101. Lanes: 1 and 2, E. coli HB101 plus vector pSE420; 3 and 4, clone 6 (pGFP-181CTPB); 5 and 6, clone 6-1 (pGFP-181CTPB plus pRUW4); 7 and 8, clone 7-1 (pGFP-181CTPB plus pRUW4). (B) Endochitinase-181CTPB secretion in E. coli C600. Lanes: 1 and 2, clone CHIT-9 (pCHIT-181CTPB) uninduced; 3 and 4, clone CHIT-1 (pCHIT-181CTPB) uninduced; 5 and 6, clone pCHIT9-1 (pCHIT-181CTPB plus pRUW4) uninduced; 7 and 8, clone pCHIT1-1 (pCHIT-181CTPB plus pRUW4) induced with 1 mM IPTG; 9 and 10, clone pCHIT9-1 (pCHIT-181CTPB plus pRUW4) induced with 1 mM IPTG. (C) Secretion of β-galactosidase–181CTPB and OmpC-181CTPB hybrids in E. coli C600. All clones were induced for 3 h with 1 mM IPTG. Lanes: 1 and 2, lysates of two clones (pβgal-181CTPB plus pRUW4); 3 and 4, the corresponding supernatants; 5 and 6, clone OmpC-3 (pOmpC-181CTPB plus pRUW4); 7 and 8, clone OmpC-8 (pOmpC-181CTPB plus pRUW4). The supernatant proteins loaded into the gel were previously concentrated 10-fold by TCA precipitation with respect to the lysates. (D) Control of protease B secretion in E. coli DH5α cells grown overnight in LB medium and antibiotics as required and visualized by Coomassie blue staining. Lanes: 1 and 2, lysate and supernatant of clone containing pRUW500 plus pRUW4; 3 and 4, lysate and supernatant of clone containing only pRUW500. (E) Control of protease B secretion in E. coli C600 cells grown overnight in LB medium and visualized as described for panel D. Lanes: 1, lysate; 2, supernatant of clone containing pRUW500 plus pRUW4.
Figure Legend Snippet: Expression of GFP- and endochitinase-181CTPB hybrids in E. coli . Protein hybrids were separated by SDS–12% PAGE; this was followed by Western blotting revealed with anti-protease B antibodies (diluted 1:1,000) and a second antibody (goat anti-rabbit immunoglobulin G coupled to peroxidase; diluted 1:1,000). Bacterial cultures harboring plasmids pGFP-181CTPB in E. coli HB101 cells and pCHIT-181CTPB in E. coli C600 were grown in LB medium with 100 μg of ampicillin/ml at 37°C until the OD 600 ). The gel was loaded with 25 μl. The same volume of supernatant (lanes S) was precipitated with 10% TCA, and the pellets were washed twice with acetone, dissolved, and loaded as described for lysates. Std, GIBCO-BRL Benchmark molecular size standard in panels A through C and GIBCO-BRL high-molecular-weight standard in panels D and E. (A) GFP-181CTPB secretion in E. coli HB101. Lanes: 1 and 2, E. coli HB101 plus vector pSE420; 3 and 4, clone 6 (pGFP-181CTPB); 5 and 6, clone 6-1 (pGFP-181CTPB plus pRUW4); 7 and 8, clone 7-1 (pGFP-181CTPB plus pRUW4). (B) Endochitinase-181CTPB secretion in E. coli C600. Lanes: 1 and 2, clone CHIT-9 (pCHIT-181CTPB) uninduced; 3 and 4, clone CHIT-1 (pCHIT-181CTPB) uninduced; 5 and 6, clone pCHIT9-1 (pCHIT-181CTPB plus pRUW4) uninduced; 7 and 8, clone pCHIT1-1 (pCHIT-181CTPB plus pRUW4) induced with 1 mM IPTG; 9 and 10, clone pCHIT9-1 (pCHIT-181CTPB plus pRUW4) induced with 1 mM IPTG. (C) Secretion of β-galactosidase–181CTPB and OmpC-181CTPB hybrids in E. coli C600. All clones were induced for 3 h with 1 mM IPTG. Lanes: 1 and 2, lysates of two clones (pβgal-181CTPB plus pRUW4); 3 and 4, the corresponding supernatants; 5 and 6, clone OmpC-3 (pOmpC-181CTPB plus pRUW4); 7 and 8, clone OmpC-8 (pOmpC-181CTPB plus pRUW4). The supernatant proteins loaded into the gel were previously concentrated 10-fold by TCA precipitation with respect to the lysates. (D) Control of protease B secretion in E. coli DH5α cells grown overnight in LB medium and antibiotics as required and visualized by Coomassie blue staining. Lanes: 1 and 2, lysate and supernatant of clone containing pRUW500 plus pRUW4; 3 and 4, lysate and supernatant of clone containing only pRUW500. (E) Control of protease B secretion in E. coli C600 cells grown overnight in LB medium and visualized as described for panel D. Lanes: 1, lysate; 2, supernatant of clone containing pRUW500 plus pRUW4.

Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Molecular Weight, Plasmid Preparation, Clone Assay, TCA Precipitation, Staining

30) Product Images from "Improved microscale cultivation of Pichia pastoris for clonal screening"

Article Title: Improved microscale cultivation of Pichia pastoris for clonal screening

Journal: Fungal Biology and Biotechnology

doi: 10.1186/s40694-018-0053-6

Microscale cultivation of phytase secreting P. pastoris ::pGAPZαB _appA under carbon-limited conditions. Clones selected in a batch-screening (compare Fig. 5 ) were analyzed in microscale cultivation in 0.8 mL BSM mod at pH 5.0 with 2% glycerol as batch substrate and 10% dextrin at 1300 rpm and 30 °C. 25 U/L amyloglucosidase was added after 17.7, 41.8, and 66.0 h (dashed vertical lines). a Dry cell weight concentrations calculated from the online backscatter signal; b Online measurement of dissolved oxygen concentrations; c Online measurement of pH; d Protein concentrations and phytase activities in culture supernatants. All values are corrected for evaporation. WT denotes the parental strain, P. pastoris X-33. Each clone was grown in quadruplicate, lines show mean values and error bars in d denote standard deviations
Figure Legend Snippet: Microscale cultivation of phytase secreting P. pastoris ::pGAPZαB _appA under carbon-limited conditions. Clones selected in a batch-screening (compare Fig. 5 ) were analyzed in microscale cultivation in 0.8 mL BSM mod at pH 5.0 with 2% glycerol as batch substrate and 10% dextrin at 1300 rpm and 30 °C. 25 U/L amyloglucosidase was added after 17.7, 41.8, and 66.0 h (dashed vertical lines). a Dry cell weight concentrations calculated from the online backscatter signal; b Online measurement of dissolved oxygen concentrations; c Online measurement of pH; d Protein concentrations and phytase activities in culture supernatants. All values are corrected for evaporation. WT denotes the parental strain, P. pastoris X-33. Each clone was grown in quadruplicate, lines show mean values and error bars in d denote standard deviations

Techniques Used: Clone Assay, Evaporation

Clonal screening of a library of phytase secreting P. pastoris ::pGAPZαB _appA . Clones were analyzed in microscale cultivation in BSM mod with 4% d -glucose. a Protein concentrations in culture supernatants were determined using the Bradford assay. b For selected clones labeled with asterisks in a, phytase activities were determined after buffer exchange in a fluorescence assay using an artificial substrate. WT denotes the parental strain, P. pastoris X-33. Each clone was grown in triplicate, error bars show standard deviations
Figure Legend Snippet: Clonal screening of a library of phytase secreting P. pastoris ::pGAPZαB _appA . Clones were analyzed in microscale cultivation in BSM mod with 4% d -glucose. a Protein concentrations in culture supernatants were determined using the Bradford assay. b For selected clones labeled with asterisks in a, phytase activities were determined after buffer exchange in a fluorescence assay using an artificial substrate. WT denotes the parental strain, P. pastoris X-33. Each clone was grown in triplicate, error bars show standard deviations

Techniques Used: Clone Assay, Bradford Assay, Labeling, Buffer Exchange, Fluorescence

31) Product Images from "Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway"

Article Title: Syndecan-1 mediates internalization of apoE-VLDL through a low density lipoprotein receptor-related protein (LRP)-independent, non-clathrin-mediated pathway

Journal: Lipids in Health and Disease

doi: 10.1186/1476-511X-5-23

Comparison of 125 I-apoE-VLDL binding to GM00701 and GM00701/Syn-1-HA . GM00701 or GM00701/Syn-HA cells were incubated with 125 I-apoE-VLDL (2 μg/ml) for 3 h at 4°C in the absence or presence of unlabeled apoE-VLDL (100 μg/ml). Unbound ligand was removed by rinsing, cells were solubilized with 0.1 M NaOH and subjected to scintillation counting. Values shown represent specific 125 I-apoE-VLDL binding and were obtained by subtracting amounts of 125 I-apoE-VLDL bound in the presence of unlabeled apoE-VLDL from total 125 I-apoE-VLDL binding. Error bars represent standard deviations of triplicate points.
Figure Legend Snippet: Comparison of 125 I-apoE-VLDL binding to GM00701 and GM00701/Syn-1-HA . GM00701 or GM00701/Syn-HA cells were incubated with 125 I-apoE-VLDL (2 μg/ml) for 3 h at 4°C in the absence or presence of unlabeled apoE-VLDL (100 μg/ml). Unbound ligand was removed by rinsing, cells were solubilized with 0.1 M NaOH and subjected to scintillation counting. Values shown represent specific 125 I-apoE-VLDL binding and were obtained by subtracting amounts of 125 I-apoE-VLDL bound in the presence of unlabeled apoE-VLDL from total 125 I-apoE-VLDL binding. Error bars represent standard deviations of triplicate points.

Techniques Used: Binding Assay, Incubation

Syn-1 internalizes DiI-apoE-VLDL through a lipid raft-mediated pathway . GM00701/Syn-1-HA cells were incubated at 37°C in the absence or presence of nystatin (25 μg/ml) for 30 min prior to a 60 min incubation with DiI-apoE-VLDL (upper panels) or 488-α 2 M (lower panels) (5 μg/ml). Cells were fixed and visualized by fluorescence microscopy (488-α 2 M, FITC filter set; DiI-apoE-VLDL, rhodamine filter set). Magnification, 630X.
Figure Legend Snippet: Syn-1 internalizes DiI-apoE-VLDL through a lipid raft-mediated pathway . GM00701/Syn-1-HA cells were incubated at 37°C in the absence or presence of nystatin (25 μg/ml) for 30 min prior to a 60 min incubation with DiI-apoE-VLDL (upper panels) or 488-α 2 M (lower panels) (5 μg/ml). Cells were fixed and visualized by fluorescence microscopy (488-α 2 M, FITC filter set; DiI-apoE-VLDL, rhodamine filter set). Magnification, 630X.

Techniques Used: Incubation, Fluorescence, Microscopy

125 I-α 2 M internalization, but not 125 I-apoE-VLDL, is inhibited by K + depletion of cells . GM00701/Syn-1-HA cells were treated with K + -depleted buffers (open symbols), or K + -containing serum-free media (closed symbols), as described in Methods. Cells were incubated with either 125 I-apoE-VLDL (2 μg/ml) (circles) or 125 I-α 2 M (0.5 μg/ml) (triangles) for 20 min at 37°C. Specificity of uptake was determined by a parallel incubation with either unlabeled apoE-VLDL (100 μg/ml) or unlabeled α 2 M (5 μg/ml), respectively. After the 37°C incubation, cells were chilled to 4°C and acid stripped with 50 mM glycine, pH 3, 100 mM NaCl for 5 min to remove surface bound ligand (that which was not internalized). Internalized ligand was quantitated by solubilizing cells with 0.1 N NaOH and scintillation counting.
Figure Legend Snippet: 125 I-α 2 M internalization, but not 125 I-apoE-VLDL, is inhibited by K + depletion of cells . GM00701/Syn-1-HA cells were treated with K + -depleted buffers (open symbols), or K + -containing serum-free media (closed symbols), as described in Methods. Cells were incubated with either 125 I-apoE-VLDL (2 μg/ml) (circles) or 125 I-α 2 M (0.5 μg/ml) (triangles) for 20 min at 37°C. Specificity of uptake was determined by a parallel incubation with either unlabeled apoE-VLDL (100 μg/ml) or unlabeled α 2 M (5 μg/ml), respectively. After the 37°C incubation, cells were chilled to 4°C and acid stripped with 50 mM glycine, pH 3, 100 mM NaCl for 5 min to remove surface bound ligand (that which was not internalized). Internalized ligand was quantitated by solubilizing cells with 0.1 N NaOH and scintillation counting.

Techniques Used: Incubation

125 I-α 2 M and 125 I-apoE-VLDL handling by GM00701 cells . GM00701 cells were incubated with 125 I-α 2 M (1 μg/ml) at 4°C for 3 h (A) or at 37°C for 1 h (B) in the absence or presence of recombinant human RAP-GST (50 μg/ml) or unlabeled α 2 M (10 μg/ml). (C) GM00701 and 3T3-L1 cells were incubated with 125 I-apoE-VLDL (2 μg/ml) supplemented with 3 μg/ml apoE at 4°C for 3 h in the absence (solid bars) or presence (shaded bars) of unlabeled apoE-VLDL (100 μg/ml). After the indicated incubation time, 125 I-ligand bound to cells at 4°C was quantitated by scintillation counting following extraction of cells with 0.1 M NaOH. 125 I-α 2 M degradation was quantitated following TCA precipitation as described in Methods. Error bars represent standard deviations of triplicate points. Inset, purified VLDL was separated by 7.5% SDS-PAGE and proteins were stained with Coomassie R. Molecular mass markers shown at left are in kD.
Figure Legend Snippet: 125 I-α 2 M and 125 I-apoE-VLDL handling by GM00701 cells . GM00701 cells were incubated with 125 I-α 2 M (1 μg/ml) at 4°C for 3 h (A) or at 37°C for 1 h (B) in the absence or presence of recombinant human RAP-GST (50 μg/ml) or unlabeled α 2 M (10 μg/ml). (C) GM00701 and 3T3-L1 cells were incubated with 125 I-apoE-VLDL (2 μg/ml) supplemented with 3 μg/ml apoE at 4°C for 3 h in the absence (solid bars) or presence (shaded bars) of unlabeled apoE-VLDL (100 μg/ml). After the indicated incubation time, 125 I-ligand bound to cells at 4°C was quantitated by scintillation counting following extraction of cells with 0.1 M NaOH. 125 I-α 2 M degradation was quantitated following TCA precipitation as described in Methods. Error bars represent standard deviations of triplicate points. Inset, purified VLDL was separated by 7.5% SDS-PAGE and proteins were stained with Coomassie R. Molecular mass markers shown at left are in kD.

Techniques Used: Incubation, Recombinant, TCA Precipitation, Purification, SDS Page, Staining

Expression of syndecan-1 in GM00701 cells . (A) GM00701 cells were transfected with pMH alone (lane 1) or pMH/Syn-1-HA (lane 2). Forty eight hours after transfection, total protein was extracted from cells with 1% Triton X-100 and equal amounts of protein (20 μg/lane) were separated by 7.5% SDS-PAGE, transferred to PVDF membrane and immunoblotted with anti-Syn-1 polyclonal antisera (1:1000) that was raised against a recombinant his-tagged fusion protein representing full length human Syn-1. Chemiluminescence detection was used to visualize bound antibodies. (B) GM00701 cells were transfected with pMH/Syn-1-HA and cells were selected that stably express Syn-1 (GM00701/Syn-1-HA). Cells were incubated with either TBS (lane 1), 8 M urea (lane 2), or 1% Triton X-100 (lane 3). Insoluble material was pelleted by centrifugation and soluble proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). (C) GM00701/Syn-1-HA cells were incubated without (lane 1) or with (lane 2) heparinase (1 unit/ml) for 5 h at 37°C. Total cellular proteins were extracted with 8 M urea, separated by 6% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). Asterisks indicate the 77 kD, non-glycosylated Syn-1 core protein [36, 37].
Figure Legend Snippet: Expression of syndecan-1 in GM00701 cells . (A) GM00701 cells were transfected with pMH alone (lane 1) or pMH/Syn-1-HA (lane 2). Forty eight hours after transfection, total protein was extracted from cells with 1% Triton X-100 and equal amounts of protein (20 μg/lane) were separated by 7.5% SDS-PAGE, transferred to PVDF membrane and immunoblotted with anti-Syn-1 polyclonal antisera (1:1000) that was raised against a recombinant his-tagged fusion protein representing full length human Syn-1. Chemiluminescence detection was used to visualize bound antibodies. (B) GM00701 cells were transfected with pMH/Syn-1-HA and cells were selected that stably express Syn-1 (GM00701/Syn-1-HA). Cells were incubated with either TBS (lane 1), 8 M urea (lane 2), or 1% Triton X-100 (lane 3). Insoluble material was pelleted by centrifugation and soluble proteins were separated by 7.5% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). (C) GM00701/Syn-1-HA cells were incubated without (lane 1) or with (lane 2) heparinase (1 unit/ml) for 5 h at 37°C. Total cellular proteins were extracted with 8 M urea, separated by 6% SDS-PAGE and immunoblotted with anti-Syn-1 antisera as described in (A). Asterisks indicate the 77 kD, non-glycosylated Syn-1 core protein [36, 37].

Techniques Used: Expressing, Transfection, SDS Page, Recombinant, Stable Transfection, Incubation, Centrifugation

Expression of Syn-1 restores apoE-VLDL uptake in GM00701 cells . GM00701 (panels A and C) and GM00701/Syn-1-HA (panels B and D) cells were incubated with DiI-labeled apoE-VLDL (4 μg/ml) for 3 h at 37°C. Unassociated ligand was removed by rinsing, cells were fixed with paraformaldehyde and observed by fluorescence microscopy (panels C and D, 550 nm excitation-573 nm emission). Corresponding phase contrast images are shown in panels A and B. Magnification, 630X.
Figure Legend Snippet: Expression of Syn-1 restores apoE-VLDL uptake in GM00701 cells . GM00701 (panels A and C) and GM00701/Syn-1-HA (panels B and D) cells were incubated with DiI-labeled apoE-VLDL (4 μg/ml) for 3 h at 37°C. Unassociated ligand was removed by rinsing, cells were fixed with paraformaldehyde and observed by fluorescence microscopy (panels C and D, 550 nm excitation-573 nm emission). Corresponding phase contrast images are shown in panels A and B. Magnification, 630X.

Techniques Used: Expressing, Incubation, Labeling, Fluorescence, Microscopy

Heparin, but not RAP-GST, competes for DiI-apoE-VLDL uptake by GM00701/Syn-1-HA cells . GM00701/Syn-1-HA cells were cultured on glass coverslips and incubated with DiI-labeled apoE-VLDL (4 μg/ml) in the absence (panel A) or presence of recombinant human RAP-GST (50 μg/ml, panel B) or heparin (200 μg/ml, panel C) at 37°C for 3 h. Cells were fixed and processed for fluorescence microscopy. Magnification, 630X.
Figure Legend Snippet: Heparin, but not RAP-GST, competes for DiI-apoE-VLDL uptake by GM00701/Syn-1-HA cells . GM00701/Syn-1-HA cells were cultured on glass coverslips and incubated with DiI-labeled apoE-VLDL (4 μg/ml) in the absence (panel A) or presence of recombinant human RAP-GST (50 μg/ml, panel B) or heparin (200 μg/ml, panel C) at 37°C for 3 h. Cells were fixed and processed for fluorescence microscopy. Magnification, 630X.

Techniques Used: Cell Culture, Incubation, Labeling, Recombinant, Fluorescence, Microscopy

32) Product Images from "Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use"

Article Title: Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74?sp chitinase inclusions, Cry crystals and spores for applied use

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-13-15

Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74 ∆s p with gfp . Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA - p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1 . Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1 . L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆ sp - gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆ sp - gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆ sp - gfp (primers: gfp-1, chiA74-4); lane 4, pEB chi A74∆sp- gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEB chi A74∆sp- gfp (primers: cytSTAB-1, gfp-3); lane 6, pEB chi A74∆sp- gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆ sp - gfp ; panel 2, 4Q7 - pEHchiA74∆ sp - gfp ; panel 3, E. coli- pEBchiA74∆ sp - gfp ; panel 4, 4Q7 - pEBchiA74∆ sp - gfp ; panel 5, E. coli DH5α; panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.
Figure Legend Snippet: Confirmation of the intracellular location of ChiA74∆s p in Escherichia coli and Bacillus thuringiensis 4Q7 using a chimeric construct with the green fluorescent protein (GFP) gene. (A) Schematic illustration that shows the fusion of chiA74 ∆s p with gfp . Panel 1, two constructs were developed, under regulation of the chiA74 wildtype promoter (wp) or under control of the cytA - p/ STAB-SD system. Lollipop indicates transcriptional terminator. Oligonucleotides used to make chimeric construct with gfp are shown in Table 1 . Panel 2, confirmation of chimeric constructs by PCR, primers used for amplification are showed in parenthesis and in Table 1 . L, 1 kb (kilobase) DNA Ladder (New England Biolabs); Lane 1, pEHchiA74∆ sp - gfp (primers: chiA74-1, chiA74-3); lane 2, pEHchiA74∆ sp - gfp (primers: chiA74-1, gfp-3); lane 3, pEHchiA74∆ sp - gfp (primers: gfp-1, chiA74-4); lane 4, pEB chi A74∆sp- gfp (primers: cytSTAB-1, chiA74-4); Lane 5, pEB chi A74∆sp- gfp (primers: cytSTAB-1, gfp-3); lane 6, pEB chi A74∆sp- gfp (primers: gfp-1, chiA74-4). (B) Phase contrast (left) and fluorescence micrographs (right) of recombinant strains of E. coli and B. thuringiensis strain 4Q7 expressing the chimeric protein ChiA74∆sp-GFP. Panel 1, E. coli- pEHchiA74∆ sp - gfp ; panel 2, 4Q7 - pEHchiA74∆ sp - gfp ; panel 3, E. coli- pEBchiA74∆ sp - gfp ; panel 4, 4Q7 - pEBchiA74∆ sp - gfp ; panel 5, E. coli DH5α; panel 6, 4Q7. Samples of recombinant strains of B. thuringiensis were collected at ~ 72 h.

Techniques Used: Construct, Polymerase Chain Reaction, Amplification, Fluorescence, Recombinant, Expressing

Expression of ChiA74Δsp in Escherichia coli DH5α and Bacillus thuringiensis 4Q7. (A) Schematic illustration of the strategy used to delete the secretion signal peptide-encoding sequence (shown as a rectangle inside the open reading frame) of chiA74 to generate chiA74∆ s p. Two constructs were developed, the first under regulation of wildtype promoter (wp) and the second under control of the strong cytA-p /STAB-SD promoter system. Lollipop indicates the putative transcriptional terminator site. Oligonucleotide sequences used to delete the signal peptide-coding sequence are shown in Table 1 . (B) Evaluation of endochitinase activity using solubilized intracellular proteins produced in E. coli. Panel 1: Zymogram using 4-MU-(GlcNAc) 3 for detection. Lane 1, E. coli- pEHchiA74∆s p ; lane 2, without sample; lane 3, E. coli- pEBchiA74∆s p; lane 4, E. coli DH5α . Black arrow indicates the position of ChiA74∆s p in recombinant E. coli strains . Panel 2: (a) E. coli- pEHchiA74∆s p , (b) E. coli- pEBchiA74∆s p. No endochitinase activity was observed with E. coli DH5α . (C) Phase contrast microscopy of recombinant strains of B. thuringiensis . Panel 1, 4Q7; panel 2, 4Q7 - pEHchiA74∆s p ; panel 3, 4Q7 - pEBchiA74∆s p . Black arrows indicate the positions of ChiA74∆s p inclusions. (D) Endochitinase activity determined using solubilized intracellular proteins of recombinant strains of B. thuringiensis. Panel 1: lane 1, 4Q7; lane 2, 4Q7 - pEHchiA74∆s p ; lane 3, 4Q7 - pEBchiA74∆s p. Black arrow indicates the position of ChiA74∆s p in recombinant 4Q7 strains . Panel 2: (a) 4Q7 - pEHchiA74∆s p , (b) 4Q7 - pEBchiA74∆s p . Activity of recombinant bacteria was normalized with the residual intracellular endochitinase activity of 4Q7.
Figure Legend Snippet: Expression of ChiA74Δsp in Escherichia coli DH5α and Bacillus thuringiensis 4Q7. (A) Schematic illustration of the strategy used to delete the secretion signal peptide-encoding sequence (shown as a rectangle inside the open reading frame) of chiA74 to generate chiA74∆ s p. Two constructs were developed, the first under regulation of wildtype promoter (wp) and the second under control of the strong cytA-p /STAB-SD promoter system. Lollipop indicates the putative transcriptional terminator site. Oligonucleotide sequences used to delete the signal peptide-coding sequence are shown in Table 1 . (B) Evaluation of endochitinase activity using solubilized intracellular proteins produced in E. coli. Panel 1: Zymogram using 4-MU-(GlcNAc) 3 for detection. Lane 1, E. coli- pEHchiA74∆s p ; lane 2, without sample; lane 3, E. coli- pEBchiA74∆s p; lane 4, E. coli DH5α . Black arrow indicates the position of ChiA74∆s p in recombinant E. coli strains . Panel 2: (a) E. coli- pEHchiA74∆s p , (b) E. coli- pEBchiA74∆s p. No endochitinase activity was observed with E. coli DH5α . (C) Phase contrast microscopy of recombinant strains of B. thuringiensis . Panel 1, 4Q7; panel 2, 4Q7 - pEHchiA74∆s p ; panel 3, 4Q7 - pEBchiA74∆s p . Black arrows indicate the positions of ChiA74∆s p inclusions. (D) Endochitinase activity determined using solubilized intracellular proteins of recombinant strains of B. thuringiensis. Panel 1: lane 1, 4Q7; lane 2, 4Q7 - pEHchiA74∆s p ; lane 3, 4Q7 - pEBchiA74∆s p. Black arrow indicates the position of ChiA74∆s p in recombinant 4Q7 strains . Panel 2: (a) 4Q7 - pEHchiA74∆s p , (b) 4Q7 - pEBchiA74∆s p . Activity of recombinant bacteria was normalized with the residual intracellular endochitinase activity of 4Q7.

Techniques Used: Expressing, Sequencing, Construct, Activity Assay, Produced, Recombinant, Microscopy

33) Product Images from "Modulation of Trehalose Dimycolate and Immune System by Rv0774c Protein Enhanced the Intracellular Survival of Mycobacterium smegmatis in Human Macrophages Cell Line"

Article Title: Modulation of Trehalose Dimycolate and Immune System by Rv0774c Protein Enhanced the Intracellular Survival of Mycobacterium smegmatis in Human Macrophages Cell Line

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2017.00289

(A) Viability of THP-1 macrophages cell after infection. Viability assay showed that Ms_rv0774c has little effect at MOI of 10:1 and 20:1 for 24 h of infection, while significantly decreased viability was observed at higher MOI of 30:1, 40:1, 50:1, and 48 h of infection (B) Intracellular survival of Ms_ve and Ms_rv0774c . CFU counts at different time intervals after infection of THP-1 macrophages with Ms_ve or Ms_rv0774c at MOI values 10 and 20. Data are representative of three independent biological replicates and shown as mean ± SD . Statistical analysis was assessed using student's t -test ( * P ≤ 0.05 and ** P ≤ 0.01).
Figure Legend Snippet: (A) Viability of THP-1 macrophages cell after infection. Viability assay showed that Ms_rv0774c has little effect at MOI of 10:1 and 20:1 for 24 h of infection, while significantly decreased viability was observed at higher MOI of 30:1, 40:1, 50:1, and 48 h of infection (B) Intracellular survival of Ms_ve and Ms_rv0774c . CFU counts at different time intervals after infection of THP-1 macrophages with Ms_ve or Ms_rv0774c at MOI values 10 and 20. Data are representative of three independent biological replicates and shown as mean ± SD . Statistical analysis was assessed using student's t -test ( * P ≤ 0.05 and ** P ≤ 0.01).

Techniques Used: Infection, Viability Assay, Mass Spectrometry

M. smegmatis expressing rv0774c has shown reduction in phosphorylation level of p38MAPK: After infection of activated THP-1 cells, the proteins were isolated, estimated, and immunoblotted by probing with the phosphorylated and unphosphorylated p38 antibody. Density of expressed proteins was quantified and expressed in relative change in phospho-p38 expression. Results shown as mean ± SD are the representation of three independent biological replicates. Statistical analysis was assessed using student's t -test ( ** P ≤ 0.05).
Figure Legend Snippet: M. smegmatis expressing rv0774c has shown reduction in phosphorylation level of p38MAPK: After infection of activated THP-1 cells, the proteins were isolated, estimated, and immunoblotted by probing with the phosphorylated and unphosphorylated p38 antibody. Density of expressed proteins was quantified and expressed in relative change in phospho-p38 expression. Results shown as mean ± SD are the representation of three independent biological replicates. Statistical analysis was assessed using student's t -test ( ** P ≤ 0.05).

Techniques Used: Expressing, Infection, Isolation

Ms_rv0774c manipulates the production of anti and pro-inflammatory cytokines by THP-1 macrophage cells: THP-1 macrophage cells were infected with Ms_rv0774c or Ms_ve at an MOI of 20. Culture supernatants were harvested after 24 h of infection and the concentrations of IL-10 (A) , IL-12 (B) , IFN-γ (C) , TNF-α (D) , and MCP-1 (E) were measured by ELISA. Data shown as mean ± SD and representative of three independent biological replicates. Statistical analysis was assessed using student's t -test ( ** P ≤ 0.01 and *** P ≤ 0.001).
Figure Legend Snippet: Ms_rv0774c manipulates the production of anti and pro-inflammatory cytokines by THP-1 macrophage cells: THP-1 macrophage cells were infected with Ms_rv0774c or Ms_ve at an MOI of 20. Culture supernatants were harvested after 24 h of infection and the concentrations of IL-10 (A) , IL-12 (B) , IFN-γ (C) , TNF-α (D) , and MCP-1 (E) were measured by ELISA. Data shown as mean ± SD and representative of three independent biological replicates. Statistical analysis was assessed using student's t -test ( ** P ≤ 0.01 and *** P ≤ 0.001).

Techniques Used: Mass Spectrometry, Infection, Enzyme-linked Immunosorbent Assay

IgG reactivity for serum samples of TB: LipY (A) and Rv0774c (B) . P-TB and EP-TB represent pulmonary and extra pulmonary TB. Experiments were performed three times in triplicates. Each dot represents mean OD of three individual experiments for individual patient. Statistical analysis was assessed using student's t -test.
Figure Legend Snippet: IgG reactivity for serum samples of TB: LipY (A) and Rv0774c (B) . P-TB and EP-TB represent pulmonary and extra pulmonary TB. Experiments were performed three times in triplicates. Each dot represents mean OD of three individual experiments for individual patient. Statistical analysis was assessed using student's t -test.

Techniques Used:

Effect of expression of rv0774c on colony size, morphology, and its growth: colony size of the M. smegmatis expressing rv0774c ( Ms_rv0774c ) (A) and containing vector alone ( Ms_ve ) (B) . (C) Average diameter (mm) reflecting the size of Ms_rv0774c and Ms_ve on day 5 of incubation at 37°C. Colonies morphology (D) Ms_rv0774c (E) Ms_ve . Colony images were digitally captured by Phase Contrast Microscope (Olympus, USA). (F) Effect of rv0774c expression on growth curve of M. smegmatis : Ms_rv0774c has shown rapid growth in lag phase as compared to Ms_ve . Each dot represents mean OD of three individual experiments.
Figure Legend Snippet: Effect of expression of rv0774c on colony size, morphology, and its growth: colony size of the M. smegmatis expressing rv0774c ( Ms_rv0774c ) (A) and containing vector alone ( Ms_ve ) (B) . (C) Average diameter (mm) reflecting the size of Ms_rv0774c and Ms_ve on day 5 of incubation at 37°C. Colonies morphology (D) Ms_rv0774c (E) Ms_ve . Colony images were digitally captured by Phase Contrast Microscope (Olympus, USA). (F) Effect of rv0774c expression on growth curve of M. smegmatis : Ms_rv0774c has shown rapid growth in lag phase as compared to Ms_ve . Each dot represents mean OD of three individual experiments.

Techniques Used: Expressing, Mass Spectrometry, Plasmid Preparation, Incubation, Microscopy

Effect of Rv0774c on drug susceptibility of M. smegmatis : (A) Mid log-phase M. smegmatis culture diluted in 7H9 medium without tween 80 and were treated with indicated concentrations of streptomycin. Resazurin dye in 20% tween 80 was added after 3 h of incubation with streptomycin, and the color change was monitored after 24 h. Pink color indicates live bacteria, while blue indicates dead bacteria (B) Survival of Ms_rv0774c and Ms_ve was monitored by counting the CFU/mL after treatment of streptomycin. Results were expressed in % survival (CFU counts without drug treatment was considered to be the 100% survival). Data are representative of three independent biological replicates and shown as mean ± SD . Statistical analysis was assessed using student's t -test ( *** P ≤ 0.001).
Figure Legend Snippet: Effect of Rv0774c on drug susceptibility of M. smegmatis : (A) Mid log-phase M. smegmatis culture diluted in 7H9 medium without tween 80 and were treated with indicated concentrations of streptomycin. Resazurin dye in 20% tween 80 was added after 3 h of incubation with streptomycin, and the color change was monitored after 24 h. Pink color indicates live bacteria, while blue indicates dead bacteria (B) Survival of Ms_rv0774c and Ms_ve was monitored by counting the CFU/mL after treatment of streptomycin. Results were expressed in % survival (CFU counts without drug treatment was considered to be the 100% survival). Data are representative of three independent biological replicates and shown as mean ± SD . Statistical analysis was assessed using student's t -test ( *** P ≤ 0.001).

Techniques Used: Incubation, Mass Spectrometry

Modulation of mycolipids in Ms_rv0774c : Quantitative estimation of (A) total lipids, (B) polar and apolar lipids extracted from the equal weight of Ms_ve or Ms_rv0774c . (C) Mycolic acid containing glycolipids were separated from the pool of total polar lipids using CHCl 3 :CH 3 OH:NH 4 OH (80:20:2) as mobile phase. (D) TLC results were expressed in term of % relative density of myco-glycolipids measured by Image J programme. Given data are expressed in mean ± SD performed in two independent experiments. Statistical analysis was assessed using student's t -test ( * P ≤ 0.05).
Figure Legend Snippet: Modulation of mycolipids in Ms_rv0774c : Quantitative estimation of (A) total lipids, (B) polar and apolar lipids extracted from the equal weight of Ms_ve or Ms_rv0774c . (C) Mycolic acid containing glycolipids were separated from the pool of total polar lipids using CHCl 3 :CH 3 OH:NH 4 OH (80:20:2) as mobile phase. (D) TLC results were expressed in term of % relative density of myco-glycolipids measured by Image J programme. Given data are expressed in mean ± SD performed in two independent experiments. Statistical analysis was assessed using student's t -test ( * P ≤ 0.05).

Techniques Used: Mass Spectrometry, Thin Layer Chromatography

(A) Measurement of NO secretion in THP-1 cells after infections of macrophages with Ms_ve or Ms_rv0774c for 24 h. (B) Transcript level of iNOS after infections was determined by qPCR. (C) The expression of TLR2 transcript was examined by real time PCR using specific primers after infection. (D) Expression of TLR2 was determined by western blot. Total proteins were isolated from THP-1 cells after infection. After electrophoresis the proteins were transferred onto nitrocellulose membrane and developed with antibodies against TLR2 and β-actin (internal control). Data are representative of three independent biological replicates and shown as mean ± SD . Statistical analysis was assessed using student's t -test ( * P ≤ 0.05 and ** P ≤ 0.01).
Figure Legend Snippet: (A) Measurement of NO secretion in THP-1 cells after infections of macrophages with Ms_ve or Ms_rv0774c for 24 h. (B) Transcript level of iNOS after infections was determined by qPCR. (C) The expression of TLR2 transcript was examined by real time PCR using specific primers after infection. (D) Expression of TLR2 was determined by western blot. Total proteins were isolated from THP-1 cells after infection. After electrophoresis the proteins were transferred onto nitrocellulose membrane and developed with antibodies against TLR2 and β-actin (internal control). Data are representative of three independent biological replicates and shown as mean ± SD . Statistical analysis was assessed using student's t -test ( * P ≤ 0.05 and ** P ≤ 0.01).

Techniques Used: Mass Spectrometry, Real-time Polymerase Chain Reaction, Expressing, Infection, Western Blot, Isolation, Electrophoresis

Green fluorescent protein expressed at 3′-end of rv0774c gene: (A) Image demonstrating the M. smegmatis expressing Rv0774c and GFP at c-terminal showing green fluorescence (B) Image taken without green fluorescence filter. (C) Expression of GFP in culture filtrate was measured by spectrofluorimetry. Green line represent fluorescence emission of culture filtrate protein of Rv0774c-GFP clone and gray line represent the GFP clone in vector alone.
Figure Legend Snippet: Green fluorescent protein expressed at 3′-end of rv0774c gene: (A) Image demonstrating the M. smegmatis expressing Rv0774c and GFP at c-terminal showing green fluorescence (B) Image taken without green fluorescence filter. (C) Expression of GFP in culture filtrate was measured by spectrofluorimetry. Green line represent fluorescence emission of culture filtrate protein of Rv0774c-GFP clone and gray line represent the GFP clone in vector alone.

Techniques Used: Expressing, Fluorescence, Plasmid Preparation

34) Product Images from "Expression of NS3/NS4A Proteins of Hepatitis C Virus in Huh7 Cells Following Engineering Its Eukaryotic Expression Vector"

Article Title: Expression of NS3/NS4A Proteins of Hepatitis C Virus in Huh7 Cells Following Engineering Its Eukaryotic Expression Vector

Journal: Jundishapur Journal of Microbiology

doi: 10.5812/jjm.27355

Polymerase Chain Reaction and Restriction Enzyme Analysis A: PCR amplification of NS3/NS4A using specific primers, 1 kb DNA ladder (Vivantis, Malaysia). B: recombinant T/A cloning vector digested by BglII and SacII enzymes, showing the 2055 bp insert, 1 kb DNA ladder (Fermentas, Lithuania). C: pDisplay-NS3/NS4A plasmid digested by BglII and SacII enzymes, showing the 2055 bp insert, 1 kb DNA ladder (Fermentas, Lithuania).
Figure Legend Snippet: Polymerase Chain Reaction and Restriction Enzyme Analysis A: PCR amplification of NS3/NS4A using specific primers, 1 kb DNA ladder (Vivantis, Malaysia). B: recombinant T/A cloning vector digested by BglII and SacII enzymes, showing the 2055 bp insert, 1 kb DNA ladder (Fermentas, Lithuania). C: pDisplay-NS3/NS4A plasmid digested by BglII and SacII enzymes, showing the 2055 bp insert, 1 kb DNA ladder (Fermentas, Lithuania).

Techniques Used: Polymerase Chain Reaction, Amplification, Recombinant, Clone Assay, Plasmid Preparation

35) Product Images from "Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group"

Article Title: Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2014.00637

Stability of plasmid pB10 and Escherichia coli DH5α in three river sediment microcosms (A–C) . Numbers indicate rate of disappearance (expressed in log per day) calculated from an exponential curve fit of the data (dotted lines). Each data point is an average of three values obtained from triplicate qPCR reactions, error bars represent standard deviation. Part of the data were presented previously in Bonot and Merlin (2010) .
Figure Legend Snippet: Stability of plasmid pB10 and Escherichia coli DH5α in three river sediment microcosms (A–C) . Numbers indicate rate of disappearance (expressed in log per day) calculated from an exponential curve fit of the data (dotted lines). Each data point is an average of three values obtained from triplicate qPCR reactions, error bars represent standard deviation. Part of the data were presented previously in Bonot and Merlin (2010) .

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

Dissemination of plasmid pB10 (A) and relative abundance of IncP-1α/β plasmids (B) in various environmental communities maintained in microcosms. The ability of each environmental matrix to support the dissemination of plasmid pB10 was deduced from the comparison of pB10 and E. coli DH5α DNA relative stabilities in microcosms overtime (rate of disappearance), as exemplified in Figure 1 for sediments A–C. Transfer of pB10 was considered as effective (+) when pB10 stability appeared significantly greater than the stability of its initial donor host. Close rate of disappearance for both pB10 and E. coli DH5α reflects an absence of transfer (-). Values between brackets represent the number of the corresponding WWTPs. effluents [#1] were sampled twice in May 2011(#1A) and February 2012 (#1B). Each data point is an average of 3 values obtained from triplicate qPCR, where error bars represent standard deviation.
Figure Legend Snippet: Dissemination of plasmid pB10 (A) and relative abundance of IncP-1α/β plasmids (B) in various environmental communities maintained in microcosms. The ability of each environmental matrix to support the dissemination of plasmid pB10 was deduced from the comparison of pB10 and E. coli DH5α DNA relative stabilities in microcosms overtime (rate of disappearance), as exemplified in Figure 1 for sediments A–C. Transfer of pB10 was considered as effective (+) when pB10 stability appeared significantly greater than the stability of its initial donor host. Close rate of disappearance for both pB10 and E. coli DH5α reflects an absence of transfer (-). Values between brackets represent the number of the corresponding WWTPs. effluents [#1] were sampled twice in May 2011(#1A) and February 2012 (#1B). Each data point is an average of 3 values obtained from triplicate qPCR, where error bars represent standard deviation.

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

36) Product Images from "Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris"

Article Title: Surface Display and Bioactivity of Bombyx mori Acetylcholinesterase on Pichia pastoris

Journal: PLoS ONE

doi: 10.1371/journal.pone.0070451

P. pastoris display of Bm AChE on an MM-B plate: 1, GS115; 2, GS115/pPIC9K- bmace-AGα1 ; 3–10, GS115/pPIC9K- bmace-AGα1 inhibited by different concentrations of eserine (clone 3: 10 −2 M, clone 4: 10 −3 M, clone 5: 10 −4 M, clone 6: 10 −5 M, clone 7: 10 −6 M, clone 8: 10 −7 M, clone 9: 10 −8 M, clone 10: 10 −9 M).
Figure Legend Snippet: P. pastoris display of Bm AChE on an MM-B plate: 1, GS115; 2, GS115/pPIC9K- bmace-AGα1 ; 3–10, GS115/pPIC9K- bmace-AGα1 inhibited by different concentrations of eserine (clone 3: 10 −2 M, clone 4: 10 −3 M, clone 5: 10 −4 M, clone 6: 10 −5 M, clone 7: 10 −6 M, clone 8: 10 −7 M, clone 9: 10 −8 M, clone 10: 10 −9 M).

Techniques Used:

Fluorescence microscopy assay of recombinant P. pastoris cells displaying Bm AChE: The fluorescence at 519 nm emitted with excitation at 495 nm was observed by fluorescence microscopy. (a) and (c), phase micrographs of recombinant yeast cells; (b) and (d), fluorescent micrographs of recombinant yeast cells. GS115/pPIC9K- bmace-AGα1 (a, b); GS115 as a control (c, d).
Figure Legend Snippet: Fluorescence microscopy assay of recombinant P. pastoris cells displaying Bm AChE: The fluorescence at 519 nm emitted with excitation at 495 nm was observed by fluorescence microscopy. (a) and (c), phase micrographs of recombinant yeast cells; (b) and (d), fluorescent micrographs of recombinant yeast cells. GS115/pPIC9K- bmace-AGα1 (a, b); GS115 as a control (c, d).

Techniques Used: Fluorescence, Microscopy, Recombinant

37) Product Images from "Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation"

Article Title: Polymer Transfected Primary Myoblasts Mediated Efficient Gene Expression and Angiogenic Proliferation

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2009.09.021

Confocal images of GFP expression from polymer transfected primary myoblasts (× 200). Control: non-treated primary myoblasts. Positive control: bPEI (25kDa)/pCMV-GFP at w/w ratio of 0.6:1. a–d: poly(CBA-DAH)/pCMV-GFP transfected primary
Figure Legend Snippet: Confocal images of GFP expression from polymer transfected primary myoblasts (× 200). Control: non-treated primary myoblasts. Positive control: bPEI (25kDa)/pCMV-GFP at w/w ratio of 0.6:1. a–d: poly(CBA-DAH)/pCMV-GFP transfected primary

Techniques Used: Expressing, Transfection, Positive Control

38) Product Images from "Identification and Functional Characterization of Sugarcane Invertase Inhibitor (ShINH1): A Potential Candidate for Reducing Pre- and Post-harvest Loss of Sucrose in Sugarcane"

Article Title: Identification and Functional Characterization of Sugarcane Invertase Inhibitor (ShINH1): A Potential Candidate for Reducing Pre- and Post-harvest Loss of Sucrose in Sugarcane

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2018.00598

Recombinant ShINH1 inhibits invertase in vitro . The activity of commercial acid invertase (Sigma) was measured after pre-incubation with 0–500 nM recombinant ShINH1 and the concentration of ShINH1 that yields 50% inhibition calculated as the IC 50 using GraphPad Prism 7.04 software. Data are presented as mean ± SD of three reactions. Statistical significance was compared between the different ShINH1 concentrations (Duncan's multiple range test; P
Figure Legend Snippet: Recombinant ShINH1 inhibits invertase in vitro . The activity of commercial acid invertase (Sigma) was measured after pre-incubation with 0–500 nM recombinant ShINH1 and the concentration of ShINH1 that yields 50% inhibition calculated as the IC 50 using GraphPad Prism 7.04 software. Data are presented as mean ± SD of three reactions. Statistical significance was compared between the different ShINH1 concentrations (Duncan's multiple range test; P

Techniques Used: Recombinant, In Vitro, Activity Assay, Incubation, Concentration Assay, Inhibition, Software

Subcellular localization of ShINH1 protein. (A) GFP-ShINH1 construct under control of the maize UBi1promoter. (B) Transient expression of UBi1GFP (control) and Ubi1GFP-ShINH1. Particle bombardment into wheat leaves and onion epidermal cells was performed and cells visualized using fluorescence and confocal microscopy 36 h after incubation at room temperature. (a and b) fluorescent and confocal images of wheat leaf cells. (c) Florescent image of onion epidermal cells. (I, III, and V: GFP controls) GFP accumulation in cytoplasm, nucleus and cell wall. (IV) Accumulation of GFP-ShINH1 in the cell wall, nucleus and cytoplasmic strands in wheat leaf cells. (II and VI) Enhanced fluorescence at the periphery of the cell indicates that GFP-ShINH1 localizes to the cell wall/apoplasmic region.
Figure Legend Snippet: Subcellular localization of ShINH1 protein. (A) GFP-ShINH1 construct under control of the maize UBi1promoter. (B) Transient expression of UBi1GFP (control) and Ubi1GFP-ShINH1. Particle bombardment into wheat leaves and onion epidermal cells was performed and cells visualized using fluorescence and confocal microscopy 36 h after incubation at room temperature. (a and b) fluorescent and confocal images of wheat leaf cells. (c) Florescent image of onion epidermal cells. (I, III, and V: GFP controls) GFP accumulation in cytoplasm, nucleus and cell wall. (IV) Accumulation of GFP-ShINH1 in the cell wall, nucleus and cytoplasmic strands in wheat leaf cells. (II and VI) Enhanced fluorescence at the periphery of the cell indicates that GFP-ShINH1 localizes to the cell wall/apoplasmic region.

Techniques Used: Construct, Expressing, Fluorescence, Confocal Microscopy, Incubation

Relative expression levels of ShINH1 in sugarcane. (A) Tissue specific expression of ShINH1 in leaf, stalk, flower, and root tissues obtained from mature sugarcane plants. Significantly increased expression of ShINH1 was observed in leaf and stalk as compared to flower and root. (B) Developmental expression of ShINH1 in sugarcane stem during sucrose accumulation. Analysis of internodes I4 (young), I8 (moderately mature) and I13 (fully mature) from pooled internodal tissues revealed significant expression of ShINH1 in I4 compared to I8 and I13. (C) Relative expression levels of ShINH1 during flower development. Flowers from stage 1 (young flowers), stage 2 (moderately mature) and stage 3 (fully mature flowers) were used. Values are mean ± SD of 3 biological replicates. Lower case letters with same or different alphabets indicate statistically non-significant and significant respectively (Duncan's multiple range test; P
Figure Legend Snippet: Relative expression levels of ShINH1 in sugarcane. (A) Tissue specific expression of ShINH1 in leaf, stalk, flower, and root tissues obtained from mature sugarcane plants. Significantly increased expression of ShINH1 was observed in leaf and stalk as compared to flower and root. (B) Developmental expression of ShINH1 in sugarcane stem during sucrose accumulation. Analysis of internodes I4 (young), I8 (moderately mature) and I13 (fully mature) from pooled internodal tissues revealed significant expression of ShINH1 in I4 compared to I8 and I13. (C) Relative expression levels of ShINH1 during flower development. Flowers from stage 1 (young flowers), stage 2 (moderately mature) and stage 3 (fully mature flowers) were used. Values are mean ± SD of 3 biological replicates. Lower case letters with same or different alphabets indicate statistically non-significant and significant respectively (Duncan's multiple range test; P

Techniques Used: Expressing

Purification and characterization of recombinant ShINH1. (A) Fractionation of recombinant ShINH1 using gel filtration chromatography. A HiTrap S-200 prepacked gel filtration column was used to separate TEV cleaved recombinant ShINH1 (peak 3) from uncleaved (His 6 -MBP: ShINH1 as peak 1) and His 6 -MBP (peak 2) fractions shown in the chromatogram. (B) Circular dichroism (CD) spectrum of recombinant ShINH1 obtained at 25°C.The CD spectrum contains minima at 222 and 209 nm, which are diagnostic of α-helical secondary structure.
Figure Legend Snippet: Purification and characterization of recombinant ShINH1. (A) Fractionation of recombinant ShINH1 using gel filtration chromatography. A HiTrap S-200 prepacked gel filtration column was used to separate TEV cleaved recombinant ShINH1 (peak 3) from uncleaved (His 6 -MBP: ShINH1 as peak 1) and His 6 -MBP (peak 2) fractions shown in the chromatogram. (B) Circular dichroism (CD) spectrum of recombinant ShINH1 obtained at 25°C.The CD spectrum contains minima at 222 and 209 nm, which are diagnostic of α-helical secondary structure.

Techniques Used: Purification, Recombinant, Fractionation, Filtration, Chromatography, Diagnostic Assay

39) Product Images from "Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii"

Article Title: Characterization of a temperature-responsive two component regulatory system from the Antarctic archaeon, Methanococcoides burtonii

Journal: Scientific Reports

doi: 10.1038/srep24278

Protein domains and structures predicted for LtrK and LtrR. ( a ) Schematic of LtrK and LtrR protein domains and sequence motifs drawn to scale. Protein domains identified using Pfam and NCBI BLAST (blue arrow boxes); predicted TMDs (hatched regions); H, N, G1, F, G2 and G3 blocks (white boxes) diagnostic of TCS histidine kinases 83 84 ; specific histidine residues H367 (H1), H443 (H2), H448 (H3), H502 (H4) of LtrK; specific aspartate residues D54 (D1), D55 (D2) and D98 (D3) of LtrR. ( b ) Homology model of the cytoplasmic domain of LtrK constructed using I-TASSER 78 . Only one subunit of the LtrK dimer is shown. The model with the highest confidence score best aligned with the structure of VicK (PDB 4I5S), a TCS SK from Streptococcus mutans , which has 37% sequence identity to the cytoplasmic domain of LtrK. The HisKA domain includes the α1 and α2 helices. The α1 helix contains the conserved H367 (red) and E368 (green) residues of the H block. The α4 helix (HATPase domain) contains the conserved N480 (orange) and R476 (blue) residues of the N block. A catalytic triad involved in autophosphorylation 45 46 is formed by R476 (blue), E368 (green) and N480 (orange). The α3 helix (between the HisKA and HATPase domains) contains the additional histidine residues H443 and H448 (magenta).
Figure Legend Snippet: Protein domains and structures predicted for LtrK and LtrR. ( a ) Schematic of LtrK and LtrR protein domains and sequence motifs drawn to scale. Protein domains identified using Pfam and NCBI BLAST (blue arrow boxes); predicted TMDs (hatched regions); H, N, G1, F, G2 and G3 blocks (white boxes) diagnostic of TCS histidine kinases 83 84 ; specific histidine residues H367 (H1), H443 (H2), H448 (H3), H502 (H4) of LtrK; specific aspartate residues D54 (D1), D55 (D2) and D98 (D3) of LtrR. ( b ) Homology model of the cytoplasmic domain of LtrK constructed using I-TASSER 78 . Only one subunit of the LtrK dimer is shown. The model with the highest confidence score best aligned with the structure of VicK (PDB 4I5S), a TCS SK from Streptococcus mutans , which has 37% sequence identity to the cytoplasmic domain of LtrK. The HisKA domain includes the α1 and α2 helices. The α1 helix contains the conserved H367 (red) and E368 (green) residues of the H block. The α4 helix (HATPase domain) contains the conserved N480 (orange) and R476 (blue) residues of the N block. A catalytic triad involved in autophosphorylation 45 46 is formed by R476 (blue), E368 (green) and N480 (orange). The α3 helix (between the HisKA and HATPase domains) contains the additional histidine residues H443 and H448 (magenta).

Techniques Used: Sequencing, Diagnostic Assay, Construct, Blocking Assay

40) Product Images from "RocA Truncation Underpins Hyper-Encapsulation, Carriage Longevity and Transmissibility of Serotype M18 Group A Streptococci"

Article Title: RocA Truncation Underpins Hyper-Encapsulation, Carriage Longevity and Transmissibility of Serotype M18 Group A Streptococci

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003842

RocA regulation of capsule synthesis modulates bacterial colony structure. Imaging of serotype M18 strains (A) GAS-M18, (B) GAS-M18 rocAM89 and (C) GAS-M18 hasko following overnight culture on Todd-Hewitt agar. i) Macroscopic imaging. ii)–iv)Scanning EM performed on individual colonies, each one imaged at three magnifications: ii) 3 k (white line = 2 µm), iii) 10 k (white line = 1 µm) and iv) 35 k (white line = 0.5 µm) respectively.
Figure Legend Snippet: RocA regulation of capsule synthesis modulates bacterial colony structure. Imaging of serotype M18 strains (A) GAS-M18, (B) GAS-M18 rocAM89 and (C) GAS-M18 hasko following overnight culture on Todd-Hewitt agar. i) Macroscopic imaging. ii)–iv)Scanning EM performed on individual colonies, each one imaged at three magnifications: ii) 3 k (white line = 2 µm), iii) 10 k (white line = 1 µm) and iv) 35 k (white line = 0.5 µm) respectively.

Techniques Used: Imaging

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Article Snippet: .. The full length genes of xopR , xopV , xopK , xopF , xopQ , xopU , xopX , xopZ and avrBs2 were PCR amplified from genomic DNA of wild type X . oryzae pv. oryzae strain with engineered 5’ CACC overhang were cloned individually into cloning vector pENTR/D/TOPO (Invitrogen, Carlsbad, CA, USA) to obtain plasmids pENTR-xopR , pENTR-xopV , pENTR-xopK , pENTR-xopF , pENTR-xopQ , pENTR-xopU , pENTR-xopX , pENTR-xopZ and pENTR-avrBs2 respectively. .. Positive clones in E. coli DH5α were screened using gene-specific primers and confirmed by sequencing.

Article Title:
Article Snippet: .. Full-length sequences of two sugarcane INVINH genes were amplified ( ShINH1 and ShINH2 ) from cDNA (0.5 μg) synthesized from RNA isolated from internodal tissues, using a standard PCR amplification protocol with high-fidelity Platinum Taq Polymerase (Thermo Fisher Scientific, USA) (Figure ). .. Amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), then E. coli DH5α cells transformed with recombinant plasmid were selected based on ampicillin resistance.

Article Title:
Article Snippet: .. Both, pVV16 and amplified Rv0774c product were digested with BamH1 and Hind III restriction enzymes, followed by ligation using T4-DNA ligase (Thermo scientific, India). ..

Stable Transfection:

Article Title:
Article Snippet: .. pCMV-MIR stable transfection pCMV-MIR vectors (OriGene) encoding for miR-19a or miR-19b and the empty vector were first amplified in E. coli DH5α and plasmids were extracted with PureLinkHipure Plasmid Filter Maxiprep Kit (Invitrogen). .. Subsequently, 1 µg of each vector was transfected into U937 cells using AMAXA Nucleofector (Lonza), according to supplier’s instructions.

Ligation:

Article Title:
Article Snippet: .. The PCR product along with expression vectors pPROEXHTa, pQE30, and pET32c was restriction digested with Bam HI/Xho I, Bam HI/Kpn I, and Bam HI/Sal I (Fermentas, USA) combination, respectively, and was followed by ligation to obtain PA-pPROEXHTa, PA-pQE30, and PA-pET32c recombinant plasmids. .. The recombinant plasmid PA-pPROEXHTa was transformed into E. coli DH5α , BL21-DE3, and DE3-pLysS, PA-pQE30 in E. coli M15 and XL-1 Blue, and PA-pET32c plasmid in E. coli BL21-DE3 and DE3-pLysS, respectively.

Article Title:
Article Snippet: .. Both, pVV16 and amplified Rv0774c product were digested with BamH1 and Hind III restriction enzymes, followed by ligation using T4-DNA ligase (Thermo scientific, India). ..

Mutagenesis:

Article Title:
Article Snippet: .. Site-directed mutagenesis and purification of mutant proteins Amino acid replacements for histidine residues (H367, H443, H448 and H502) in LtrK (plasmid pReceiver-B03-LtrK) and aspartate residues (D54, D55 and D98) in LtrR (plasmid pJexpress404-LtrR) were generated by site-directed mutagenesis using a Phusion Site-Directed Mutagenesis kit (Thermo Scientific) according to the manufacturer’s protocol. .. PCR amplified DNA was circularized by incubating 20 ng of PCR product with 0.5 μl of T4 DNA ligase for 5 min, and the circularized plasmid was cloned in E. coli DH5α.

Isolation:

Article Title:
Article Snippet: .. Full-length sequences of two sugarcane INVINH genes were amplified ( ShINH1 and ShINH2 ) from cDNA (0.5 μg) synthesized from RNA isolated from internodal tissues, using a standard PCR amplification protocol with high-fidelity Platinum Taq Polymerase (Thermo Fisher Scientific, USA) (Figure ). .. Amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), then E. coli DH5α cells transformed with recombinant plasmid were selected based on ampicillin resistance.

Synthesized:

Article Title:
Article Snippet: .. Full-length sequences of two sugarcane INVINH genes were amplified ( ShINH1 and ShINH2 ) from cDNA (0.5 μg) synthesized from RNA isolated from internodal tissues, using a standard PCR amplification protocol with high-fidelity Platinum Taq Polymerase (Thermo Fisher Scientific, USA) (Figure ). .. Amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), then E. coli DH5α cells transformed with recombinant plasmid were selected based on ampicillin resistance.

Generated:

Article Title:
Article Snippet: .. Site-directed mutagenesis and purification of mutant proteins Amino acid replacements for histidine residues (H367, H443, H448 and H502) in LtrK (plasmid pReceiver-B03-LtrK) and aspartate residues (D54, D55 and D98) in LtrR (plasmid pJexpress404-LtrR) were generated by site-directed mutagenesis using a Phusion Site-Directed Mutagenesis kit (Thermo Scientific) according to the manufacturer’s protocol. .. PCR amplified DNA was circularized by incubating 20 ng of PCR product with 0.5 μl of T4 DNA ligase for 5 min, and the circularized plasmid was cloned in E. coli DH5α.

Cell Culture:

Article Title:
Article Snippet: .. GAS were cultured on Columbia horse blood agar plates (OXOID) Todd-Hewitt (TH) agar or in TH broth (OXOID) at 37°C, 5% CO2 for 16 hours. .. E. coli XL-10 gold (Stratagene) and DH5α (Invitrogen) were grown in LB broth.

Purification:

Article Title:
Article Snippet: .. Site-directed mutagenesis and purification of mutant proteins Amino acid replacements for histidine residues (H367, H443, H448 and H502) in LtrK (plasmid pReceiver-B03-LtrK) and aspartate residues (D54, D55 and D98) in LtrR (plasmid pJexpress404-LtrR) were generated by site-directed mutagenesis using a Phusion Site-Directed Mutagenesis kit (Thermo Scientific) according to the manufacturer’s protocol. .. PCR amplified DNA was circularized by incubating 20 ng of PCR product with 0.5 μl of T4 DNA ligase for 5 min, and the circularized plasmid was cloned in E. coli DH5α.

Polymerase Chain Reaction:

Article Title:
Article Snippet: .. The PCR product along with expression vectors pPROEXHTa, pQE30, and pET32c was restriction digested with Bam HI/Xho I, Bam HI/Kpn I, and Bam HI/Sal I (Fermentas, USA) combination, respectively, and was followed by ligation to obtain PA-pPROEXHTa, PA-pQE30, and PA-pET32c recombinant plasmids. .. The recombinant plasmid PA-pPROEXHTa was transformed into E. coli DH5α , BL21-DE3, and DE3-pLysS, PA-pQE30 in E. coli M15 and XL-1 Blue, and PA-pET32c plasmid in E. coli BL21-DE3 and DE3-pLysS, respectively.

Article Title:
Article Snippet: .. The full length genes of xopR , xopV , xopK , xopF , xopQ , xopU , xopX , xopZ and avrBs2 were PCR amplified from genomic DNA of wild type X . oryzae pv. oryzae strain with engineered 5’ CACC overhang were cloned individually into cloning vector pENTR/D/TOPO (Invitrogen, Carlsbad, CA, USA) to obtain plasmids pENTR-xopR , pENTR-xopV , pENTR-xopK , pENTR-xopF , pENTR-xopQ , pENTR-xopU , pENTR-xopX , pENTR-xopZ and pENTR-avrBs2 respectively. .. Positive clones in E. coli DH5α were screened using gene-specific primers and confirmed by sequencing.

Article Title:
Article Snippet: .. Full-length sequences of two sugarcane INVINH genes were amplified ( ShINH1 and ShINH2 ) from cDNA (0.5 μg) synthesized from RNA isolated from internodal tissues, using a standard PCR amplification protocol with high-fidelity Platinum Taq Polymerase (Thermo Fisher Scientific, USA) (Figure ). .. Amplified products were cloned into the pGEM-T Easy vector (Promega, Madison, WI, USA), then E. coli DH5α cells transformed with recombinant plasmid were selected based on ampicillin resistance.

Random Hexamer Labeling:

Article Title:
Article Snippet: .. To convert the total RNA into labeled cDNA from the E. coli DH5α samples treated with FCM, ACM, or 0.5× LB, reverse transcription was performed in 1.5-ml microcentrifuge tubes (Fisher) to which 6 μg of total RNA and 6 μg of random hexamer primers (Invitrogen Corp., Carlsbad, Calif.) were added. .. The volume was adjusted to 24 μl with RNase-free water (Invitrogen).

other:

Article Title:
Article Snippet: The bacterial strains used in this study were E. coli MRE600, DH5α (Invitrogen), and BL21-DE3 (Invitrogen).

Article Title:
Article Snippet: Bacterial strains E . coli DH5α purchased from Life Technologies, genotype F− Φ80lac ZΔM15 Δ(lac ZYA-arg F) U169 rec A1 end A1 hsd R17 (rK−, mK+) pho A sup E44 λ− thi -1 gyr A96 rel A1 λ- was used for plasmids clonning and long term storage.

Article Title:
Article Snippet: To construct the yeast expression vector pPICZα-IL18WT, the mature sequence of IL-18 was sub-cloned into pPICZαA (Invitrogen) at the Eco RI and Xb aI sites and then transformed into E . coli DH5α.

Article Title:
Article Snippet: The template for in vitro transcription was prepared by PCR amplification of entire GCN4 ORF followed by cloning into pCR2.1-Topo vector (Invitrogen) according to the manufacturer's instructions.

Article Title:
Article Snippet: For transfections, GM00701 cells were plated onto 10 cm tissue culture dishes and incubated with 8 μg vector/dish together with Lipofectamine Plus according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).

Article Title:
Article Snippet: Recombinant NSP1-His and GST-NSP2 were produced in E. coli strains DH5α and BL21 and purified using the ProBond resin (Invitrogen) and glutathione-agarose (Novagen).

Article Title:
Article Snippet: See ( ) for more details] Polymerase for PCR amplification, T4 DNA ligase (restriction enzymes as needed for digestion of PCR products prior to ligation), BP Clonase enzyme mix (Invitrogen) 30°C, 37°C and 43°C incubators Competent cells of E. coli DH5α and appropriate medium for growth Eppendorf and PCR tubes, tips and pipetman PCR machine TSB and TSA to grow S. aureus Ampicillin and chloramphenicol antibiotics used at the final concentrations of 100 and 10 µg/mL, for selection of E. coli and S. aureus carrying pKOR1, respectively.

Article Title:
Article Snippet: All constructs (Figure A) were propagated in E. coli DH5α [supE44, DlacU169 (F80lacZDM15 ) hsdR17 recA1 end A1 gyrA96 thi-1 relA1 ] (Invitrogen, Carlsbad CA, USA) and then used for transforming the acrystalliferous strain of B. thuringiensis subsp. israelensis 4Q7, (hereafter 4Q7), and B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) (Bacillus Genetic Stock Center, Columbus, OH).

Article Title:
Article Snippet: Subsequent to restriction endonuclease digestion with Asc 1 and Pme 1, the product was ligated into the lentiviral expression vector, pLenti6.3-MCS/V5 DEST (Invitrogen), which was used to transform E. coli DH5α cells.

Article Title:
Article Snippet: P. aeruginosa strains were cultured in LB broth or minimal medium P (MMP) supplemented with different concentration of proline (Oxoid Ltd.) or glutamate (Oxoid Ltd.).

Article Title:
Article Snippet: Kanamycin was obtained from duchefa, Taq polymerase, 2x PCR master mixes, 1 kb DNA Ladder, NheI and XhoI Restriction Enzymes, T4 DNA Ligase, IPTG (isopropyl-β-D-thiogalactopyranoside) and Protein ladder were obtained from fermentas.

Article Title:
Article Snippet: Basically, sets of qPCR primers were designed to prime on both sides of a unique junction between building blocks of the element targeted for quantification: a unique assembly between two truncated transposons for plasmid pB10 (target coordinates 45968–46102; GenBank NC_004840), and both sides of a unique 97 kb-deletion in the chromosome of E. coli DH5α (target coordinates on K12 274654–372933; GenBank U00096; ; ). qPCRs were performed in triplicate using a “Step One Plus Real-Time PCR System” (Applied Biosystems, driver: StepOne Software v2.2) with thermocycling conditions set as follows: 2 min at 50°C, then 10 min at 95°C followed by 45 cycles of 15 s at 95°C and 1 min at 60°C.

Article Title:
Article Snippet: The gene appA (gene ID 946206) from E. coli K-12 substrain MG1655 was codon optimized for P. pastoris and synthesized by GeneArt™ (Regensburg, Germany) and inserted, after digestion with Pst I and Not I, into the vector pGAPZαB (Invitrogen).

Article Title:
Article Snippet: The PCR products were ligated into the promoterless pBlue-TOPO and pPROBE-NT vectors and transformed into E. coli strain, Top10 (Invitrogen Technologies, Carlsbad, CA) and DH5α strain, respectively [ ].

Article Title:
Article Snippet: A single colony from the transformed plates was tested by colony-PCR with specific primers for the A. baumannii Tnp gene and sequenced using an ABI Prism 3730XL Analyzer (Applied Biosystems).

Article Title:
Article Snippet: The following E . coli strains were used: strain DH5α was used as a reference for ATP synthase, strain INVαF′ (Invitrogen, Carlsbad, Calif.) was used as a host for plasmids carrying PCR clones, and ATP synthase-negative (Δ unc ) mutant DK8 ( ) (obtained from M. Futai, Osaka University, Osaka, Japan) was used as a host to screen the genomic library of C . pasteurianum in plasmid pBR322.

Article Title:
Article Snippet: Membrane permeabilization assays The effect of SP-A on the cell membrane integrity of P. aeruginosa and E. coli DH5α was assessed by determining permeability to a phosphatase substrate, Enzyme-Labeled Fluorescence 97 (ELF-97) (Molecular Probes, Carlsbad, CA), as we have previously described , , . hSP-A (50 µg/ml) or mouse BAL fluids (50 µg total protein) was incubated with 1×108 stationary phase P. aeruginosa or E. coli bacteria/ml in 100 µl of membrane permeabilization buffer for 15 min at 37°C, and 100 µM ELF97 phosphatase substrate was added.

Article Title:
Article Snippet: Bacterial Strains Escherichia coli strain BL21 (DE3) pLys (Novagen, Madison, WI, USA) was used as the host strain for expression of recombinant multi-epitope protein and E. coli DH5α (Invitrogen, San Diego, CA, USA) was used for obtaining plasmids.

Article Title:
Article Snippet: E. coli JM105 and DH5α were from Pharmacia, and TOP10 was from Invitrogen.

Article Title:
Article Snippet: The lnc-CC3 over-expression plasmid was transfected into SiHa cells with Lipofectamine 2000 (Invitrogen, Carlsbad, USA); pcDNA3.1 (+) vector transfected SiHa cells were used as controls.

Article Title:
Article Snippet: The P. pastoris GS115 strains and the integrative expression vector (pPIC9K) were obtained from Invitrogen Biotechnology Co. (Shanghai, China).

Article Title:
Article Snippet: PpiA overexpression in L. lactis ppiA ORF (including RBS sequence) from IL1403 strain (GenBank Accession number AAK04463.1) was cloned on an expression vector to avoid, when overexpressed in MG1363 strain, any recombination with the chromosomal gene copy. ppiA ORF was PCR-amplified from IL1403 genomic DNA using ppiARBS and ppiATer primers , and the resulting ppiA fragment (961 bp) was ligated into pCR2.1-TOPO (Invitrogen).

Article Title:
Article Snippet: DNA was amplified using the linker as a primer to generate a sufficient amount to clone into the pCR II-TOPO vector using TOPO TA Cloning Kit with One Shot Max Efficiency DH5α-T1 E. coli according to the manufacturer's suggested protocol (Invitrogen).

Article Title:
Article Snippet: The plasmids, pCMV-Luc, pCMV-GFP, and pCMV-VEGF165 , containing a firefly luciferase reporter gene, green fluorescent protein gene and vascular endothelial growth factor gene 165, respectively, were amplified in E. coli DH5α and purified by standard Maxiprep kit (Invitrogen, Carlsbad, CA), separately.

Article Title:
Article Snippet: The primers were used for amplification (GeneAmp PCR system 9600; Perkin-Elmer Corp., Norwalk, Conn.) of purified chromosomal DNA from B. pilosicoli strain SP16 in a total volume of 75 μl containing 4 mM MgCl2 ; 1× PCR buffer; 0.2 mM (each) dATP, dCTP, dGTP, and dTTP; a 1-μM concentration of each primer; and 1.5 U of Taq DNA polymerase (GIBCO-BRL) in sterile filtered autoclaved water.

Labeling:

Article Title:
Article Snippet: .. To convert the total RNA into labeled cDNA from the E. coli DH5α samples treated with FCM, ACM, or 0.5× LB, reverse transcription was performed in 1.5-ml microcentrifuge tubes (Fisher) to which 6 μg of total RNA and 6 μg of random hexamer primers (Invitrogen Corp., Carlsbad, Calif.) were added. .. The volume was adjusted to 24 μl with RNase-free water (Invitrogen).

Expressing:

Article Title:
Article Snippet: .. The respective recombinant plasmid was extracted and cleaved by BglII and SacII (Fermentas, Lithuania) and inserted into the similarly digested eukaryotic expression vector pDisplay (Invitrogen, Carlsbad, CA, USA) with T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) and transformed into E. coli DH5α. .. The pDisplay vector contains hemagglutinin A (HA) epitope tag in upstream and myc epitope in downstream of the cut sites which allow for the detection of the expressed recombinant proteins by immunofluorescence assay using anti-HA/myc antibodies.

Article Title:
Article Snippet: .. The PCR product along with expression vectors pPROEXHTa, pQE30, and pET32c was restriction digested with Bam HI/Xho I, Bam HI/Kpn I, and Bam HI/Sal I (Fermentas, USA) combination, respectively, and was followed by ligation to obtain PA-pPROEXHTa, PA-pQE30, and PA-pET32c recombinant plasmids. .. The recombinant plasmid PA-pPROEXHTa was transformed into E. coli DH5α , BL21-DE3, and DE3-pLysS, PA-pQE30 in E. coli M15 and XL-1 Blue, and PA-pET32c plasmid in E. coli BL21-DE3 and DE3-pLysS, respectively.

Article Title:
Article Snippet: .. Plasmid pMV261 (for over-expression) and/or pACT (an acetamide-inducible expression vector, for reduced expression) (Invitrogen, Waltham, MA, United States) were digested with the same restriction enzymes. .. The two fragments were ligated, transformed into E. coli DH5α and plated on LB agar containing kanamycin (50 μg/mL).

Sequencing:

Article Title:
Article Snippet: .. After sequencing to confirm their identity, RDR1 and RDR6 clones were used to generate recombinant vectors for VIGS as described by Liu and colleagues [ ], using the Gateway technology (Invitrogen, Carlsbad, CA) and att B primer pairs for BP clonase. .. The cDNA fragments were subsequently transferred to the destination vector pTRV2 by LR clonase and used to transform E . coli DH5α competent cells.

Transformation Assay:

Article Title:
Article Snippet: .. The respective recombinant plasmid was extracted and cleaved by BglII and SacII (Fermentas, Lithuania) and inserted into the similarly digested eukaryotic expression vector pDisplay (Invitrogen, Carlsbad, CA, USA) with T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) and transformed into E. coli DH5α. .. The pDisplay vector contains hemagglutinin A (HA) epitope tag in upstream and myc epitope in downstream of the cut sites which allow for the detection of the expressed recombinant proteins by immunofluorescence assay using anti-HA/myc antibodies.

Article Title:
Article Snippet: .. Development of Binary Vectors and Genetic Transformation of A. annua The ORF of AaHDR1 with its terminal codon was cloned to the pENTY/D-TOPO vector (Gateway, Invitrogen) to obtain a recombinant pENTY-AaHDR1 plasmid, which was then introduced to competent cells of E. coli DH5α. .. Given that pENTY/D-TOPO vector can allow both sense and anti-sense orientation ligation, 10 positive individual colonies were selected to isolate plasmids for sequencing to identify sense or anti-sense insertion orientations of the AaHDR1 ORF.

Recombinant:

Article Title:
Article Snippet: .. The respective recombinant plasmid was extracted and cleaved by BglII and SacII (Fermentas, Lithuania) and inserted into the similarly digested eukaryotic expression vector pDisplay (Invitrogen, Carlsbad, CA, USA) with T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) and transformed into E. coli DH5α. .. The pDisplay vector contains hemagglutinin A (HA) epitope tag in upstream and myc epitope in downstream of the cut sites which allow for the detection of the expressed recombinant proteins by immunofluorescence assay using anti-HA/myc antibodies.

Article Title:
Article Snippet: .. The PCR product along with expression vectors pPROEXHTa, pQE30, and pET32c was restriction digested with Bam HI/Xho I, Bam HI/Kpn I, and Bam HI/Sal I (Fermentas, USA) combination, respectively, and was followed by ligation to obtain PA-pPROEXHTa, PA-pQE30, and PA-pET32c recombinant plasmids. .. The recombinant plasmid PA-pPROEXHTa was transformed into E. coli DH5α , BL21-DE3, and DE3-pLysS, PA-pQE30 in E. coli M15 and XL-1 Blue, and PA-pET32c plasmid in E. coli BL21-DE3 and DE3-pLysS, respectively.

Article Title:
Article Snippet: .. After sequencing to confirm their identity, RDR1 and RDR6 clones were used to generate recombinant vectors for VIGS as described by Liu and colleagues [ ], using the Gateway technology (Invitrogen, Carlsbad, CA) and att B primer pairs for BP clonase. .. The cDNA fragments were subsequently transferred to the destination vector pTRV2 by LR clonase and used to transform E . coli DH5α competent cells.

Article Title:
Article Snippet: .. Development of Binary Vectors and Genetic Transformation of A. annua The ORF of AaHDR1 with its terminal codon was cloned to the pENTY/D-TOPO vector (Gateway, Invitrogen) to obtain a recombinant pENTY-AaHDR1 plasmid, which was then introduced to competent cells of E. coli DH5α. .. Given that pENTY/D-TOPO vector can allow both sense and anti-sense orientation ligation, 10 positive individual colonies were selected to isolate plasmids for sequencing to identify sense or anti-sense insertion orientations of the AaHDR1 ORF.

Over Expression:

Article Title:
Article Snippet: .. Plasmid pMV261 (for over-expression) and/or pACT (an acetamide-inducible expression vector, for reduced expression) (Invitrogen, Waltham, MA, United States) were digested with the same restriction enzymes. .. The two fragments were ligated, transformed into E. coli DH5α and plated on LB agar containing kanamycin (50 μg/mL).

Plasmid Preparation:

Article Title:
Article Snippet: .. The respective recombinant plasmid was extracted and cleaved by BglII and SacII (Fermentas, Lithuania) and inserted into the similarly digested eukaryotic expression vector pDisplay (Invitrogen, Carlsbad, CA, USA) with T4 DNA ligase (Invitrogen, Carlsbad, CA, USA) and transformed into E. coli DH5α. .. The pDisplay vector contains hemagglutinin A (HA) epitope tag in upstream and myc epitope in downstream of the cut sites which allow for the detection of the expressed recombinant proteins by immunofluorescence assay using anti-HA/myc antibodies.

Article Title:
Article Snippet: .. Site-directed mutagenesis and purification of mutant proteins Amino acid replacements for histidine residues (H367, H443, H448 and H502) in LtrK (plasmid pReceiver-B03-LtrK) and aspartate residues (D54, D55 and D98) in LtrR (plasmid pJexpress404-LtrR) were generated by site-directed mutagenesis using a Phusion Site-Directed Mutagenesis kit (Thermo Scientific) according to the manufacturer’s protocol. .. PCR amplified DNA was circularized by incubating 20 ng of PCR product with 0.5 μl of T4 DNA ligase for 5 min, and the circularized plasmid was cloned in E. coli DH5α.

Article Title:
Article Snippet: .. pCMV-MIR stable transfection pCMV-MIR vectors (OriGene) encoding for miR-19a or miR-19b and the empty vector were first amplified in E. coli DH5α and plasmids were extracted with PureLinkHipure Plasmid Filter Maxiprep Kit (Invitrogen). .. Subsequently, 1 µg of each vector was transfected into U937 cells using AMAXA Nucleofector (Lonza), according to supplier’s instructions.

Article Title:
Article Snippet: .. Development of Binary Vectors and Genetic Transformation of A. annua The ORF of AaHDR1 with its terminal codon was cloned to the pENTY/D-TOPO vector (Gateway, Invitrogen) to obtain a recombinant pENTY-AaHDR1 plasmid, which was then introduced to competent cells of E. coli DH5α. .. Given that pENTY/D-TOPO vector can allow both sense and anti-sense orientation ligation, 10 positive individual colonies were selected to isolate plasmids for sequencing to identify sense or anti-sense insertion orientations of the AaHDR1 ORF.

Article Title:
Article Snippet: .. The full length genes of xopR , xopV , xopK , xopF , xopQ , xopU , xopX , xopZ and avrBs2 were PCR amplified from genomic DNA of wild type X . oryzae pv. oryzae strain with engineered 5’ CACC overhang were cloned individually into cloning vector pENTR/D/TOPO (Invitrogen, Carlsbad, CA, USA) to obtain plasmids pENTR-xopR , pENTR-xopV , pENTR-xopK , pENTR-xopF , pENTR-xopQ , pENTR-xopU , pENTR-xopX , pENTR-xopZ and pENTR-avrBs2 respectively. .. Positive clones in E. coli DH5α were screened using gene-specific primers and confirmed by sequencing.

Article Title:
Article Snippet: .. Expressions of CYP98A3 with the pCWori vector in BL21 (DE3) and XL-Blue cells (both from Invitrogen) in rich media yielded lower P450 levels than obtained in DH5α cells. ..

Article Title:
Article Snippet: .. Plasmid pMV261 (for over-expression) and/or pACT (an acetamide-inducible expression vector, for reduced expression) (Invitrogen, Waltham, MA, United States) were digested with the same restriction enzymes. .. The two fragments were ligated, transformed into E. coli DH5α and plated on LB agar containing kanamycin (50 μg/mL).

Article Title:
Article Snippet: .. Approximately 100 ng of the vector together with inserts (~2 times molar excess of each) were used in the 20 μl CPEC reaction with Phusion High-Fidelity DNA Polymerase (Life Technologies, US). .. Five microliters from the reaction were used for the transformation of competent TOP10 F’ or DH5α strain E. coli cells.

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    Thermo Fisher e coli dh5α
    PCR amplification of E.coli <t>DH5α</t> clones Lane 2 to 7 –positive amplicons Lane1- DNA marker
    E Coli Dh5α, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli dh5α/product/Thermo Fisher
    Average 93 stars, based on 343 article reviews
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    e coli dh5α - by Bioz Stars, 2020-05
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    93
    Thermo Fisher luciferase expressing e coli dh5α paklux2
    ICE Mh1 PM22 fitness costs, addiction, and consequences for antimicrobial resistance in transconjugants. (A) Growth curves of P. multocida CCUG 17976 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (B) Growth curves of M. haemolytica ATCC 33396 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (C) Luciferase-based competition index from co-cultures of ICE Mh1 PM22 transconjugants and E. coli <t>DH5α</t> harboring the luciferase expression plasmid <t>pAKlux2.</t> Mean of 4 biological replicates with SEM; t-test, ∗ P ≤ 0.05. (D) Long-term repeated passage of ICE Mh1 PM22 transconjugants. Transconjugants were grown in MH (no drug) or MH + 0.5 MIC (subinhibitory; MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396 WT) for 150 days and plated on media with or without selective concentrations of indicated antimicrobials (i.e., 0.5 MIC for non-susceptible isogenic transconjugants). Mean of 3 biological replicates with SEM. (E) Upper panel: growth curves of co-cultures of E. coli DH5α pAKlux2 and E. coli DH5α, P. multocida CCUG 17976 ICE Mh1 PM22 , or M. haemolytica ATCC 33396 ICE Mh1 PM22 in subinhibitory (0.5 MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396) concentrations of oxytetracycline (left) or spectinomycin (right). Mean of 3 biological replicates with SEM. Lower panel: detection of E. coli luciferase production in co-cultures (as above) with either oxytetracycline (left) or spectinomycin (right). (F) Schematic of checkerboard synergy assay for drug interaction screening.
    Luciferase Expressing E Coli Dh5α Paklux2, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/luciferase expressing e coli dh5α paklux2/product/Thermo Fisher
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    PCR amplification of E.coli DH5α clones Lane 2 to 7 –positive amplicons Lane1- DNA marker

    Journal: Veterinary World

    Article Title: Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET-32a – Escherichia coli DH5α system

    doi: 10.14202/vetworld.2018.557-561

    Figure Lengend Snippet: PCR amplification of E.coli DH5α clones Lane 2 to 7 –positive amplicons Lane1- DNA marker

    Article Snippet: The pET-32a ligA plasmid construct was extracted from the transformed E. coli DH5α using Thermo Scientific Gene JET plasmid Miniprep Kit and subjected to sequencing.

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay, Marker

    PCR amplification of E.coli DH5α clones Lane 2 to 7 –positive amplicons Lane1- DNA marker

    Journal: Veterinary World

    Article Title: Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET-32a – Escherichia coli DH5α system

    doi: 10.14202/vetworld.2018.557-561

    Figure Lengend Snippet: PCR amplification of E.coli DH5α clones Lane 2 to 7 –positive amplicons Lane1- DNA marker

    Article Snippet: The pET-32a ligA plasmid construct was extracted from the transformed E. coli DH5α using Thermo Scientific Gene JET plasmid Miniprep Kit and subjected to sequencing.

    Techniques: Polymerase Chain Reaction, Amplification, Clone Assay, Marker

    ICE Mh1 PM22 fitness costs, addiction, and consequences for antimicrobial resistance in transconjugants. (A) Growth curves of P. multocida CCUG 17976 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (B) Growth curves of M. haemolytica ATCC 33396 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (C) Luciferase-based competition index from co-cultures of ICE Mh1 PM22 transconjugants and E. coli DH5α harboring the luciferase expression plasmid pAKlux2. Mean of 4 biological replicates with SEM; t-test, ∗ P ≤ 0.05. (D) Long-term repeated passage of ICE Mh1 PM22 transconjugants. Transconjugants were grown in MH (no drug) or MH + 0.5 MIC (subinhibitory; MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396 WT) for 150 days and plated on media with or without selective concentrations of indicated antimicrobials (i.e., 0.5 MIC for non-susceptible isogenic transconjugants). Mean of 3 biological replicates with SEM. (E) Upper panel: growth curves of co-cultures of E. coli DH5α pAKlux2 and E. coli DH5α, P. multocida CCUG 17976 ICE Mh1 PM22 , or M. haemolytica ATCC 33396 ICE Mh1 PM22 in subinhibitory (0.5 MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396) concentrations of oxytetracycline (left) or spectinomycin (right). Mean of 3 biological replicates with SEM. Lower panel: detection of E. coli luciferase production in co-cultures (as above) with either oxytetracycline (left) or spectinomycin (right). (F) Schematic of checkerboard synergy assay for drug interaction screening.

    Journal: Frontiers in Microbiology

    Article Title: Emerging Variants of the Integrative and Conjugant Element ICEMh1 in Livestock Pathogens: Structural Insights, Potential Host Range, and Implications for Bacterial Fitness and Antimicrobial Therapy

    doi: 10.3389/fmicb.2019.02608

    Figure Lengend Snippet: ICE Mh1 PM22 fitness costs, addiction, and consequences for antimicrobial resistance in transconjugants. (A) Growth curves of P. multocida CCUG 17976 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (B) Growth curves of M. haemolytica ATCC 33396 (spontaneous rifampin-resistant mutant) and isogenic ICE Mh1 PM22 transconjugant. Mean of 4 biological replicates with SEM. (C) Luciferase-based competition index from co-cultures of ICE Mh1 PM22 transconjugants and E. coli DH5α harboring the luciferase expression plasmid pAKlux2. Mean of 4 biological replicates with SEM; t-test, ∗ P ≤ 0.05. (D) Long-term repeated passage of ICE Mh1 PM22 transconjugants. Transconjugants were grown in MH (no drug) or MH + 0.5 MIC (subinhibitory; MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396 WT) for 150 days and plated on media with or without selective concentrations of indicated antimicrobials (i.e., 0.5 MIC for non-susceptible isogenic transconjugants). Mean of 3 biological replicates with SEM. (E) Upper panel: growth curves of co-cultures of E. coli DH5α pAKlux2 and E. coli DH5α, P. multocida CCUG 17976 ICE Mh1 PM22 , or M. haemolytica ATCC 33396 ICE Mh1 PM22 in subinhibitory (0.5 MIC for susceptible P. multocida CCUG 17976 or M. haemolytica ATCC 33396) concentrations of oxytetracycline (left) or spectinomycin (right). Mean of 3 biological replicates with SEM. Lower panel: detection of E. coli luciferase production in co-cultures (as above) with either oxytetracycline (left) or spectinomycin (right). (F) Schematic of checkerboard synergy assay for drug interaction screening.

    Article Snippet: For 2-strain co-culture experiments, luciferase-expressing E. coli DH5α pAKlux2 and test strains (including DH5α without pAKlux2 as a control) were each inoculated at an OD600 of 0.0025 (i.e., total OD600 of ∼0.005) into black clear bottom 96-well plates (Nunc, Thermo-Fisher Scientific, Ottawa, ON, Canada).

    Techniques: Mutagenesis, Luciferase, Expressing, Plasmid Preparation