Structured Review

GE Healthcare pcr buffer
RC1339/ APRc is expressed in Rickettsia conorii and Rickettsia rickettsii and accumulates at the outer membrane in both species. (A) <t>RT-PCR</t> analysis of RC1339/ APRc expression on rickettsial spp.. The housekeeping gene hrtA (17 kDa surface antigen) was used as a control. The negative control for the <t>cDNA</t> synthesis lacking reverse transcriptase is identified by (RTase -). Rickettsial species are identified on the top and the gene names are shown on the left side of the agarose gel. (B) A whole cell lysate from R. rickettsii (1) and insoluble (2) and soluble (3) fractions from R. conorii extracts were isolated and then subjected to Western Blot analysis with anti-APRc antibody. A specific band with approximately 21 kDa was detected. (C) Whole cell lysates (WCL), inner (IM) and outer membrane (OM) fractions from sarkosyl treatment of R. rickettsii and R. conorii extracts were isolated and then subjected to Western Blot analysis with anti-APRc and anti-rOmpB antibody. APRc shares the same localization of rOmpB, an internal marker for outer membrane of Rickettsia spp. Molecular mass markers in kilodaltons (kDa) are shown on the left. (D) For flow cytometric analysis of APRc expression in R. conorii , fixed bacteria were queried for deposition of anti-APRc (orange trace), negative control lacking primay antibody (blue trace), or the positive control anti-OmpB (red trace), a known rickettsial surface protein. After incubation with fluorescent secondary antibody, both anti-APRc and anti-OmpB detected on the bacterial surface (increased fluorescence), indicating accessibility of these target proteins to exogenously applied antibody.
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1) Product Images from "RC1339/APRc from Rickettsia conorii Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes"

Article Title: RC1339/APRc from Rickettsia conorii Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1004324

RC1339/ APRc is expressed in Rickettsia conorii and Rickettsia rickettsii and accumulates at the outer membrane in both species. (A) RT-PCR analysis of RC1339/ APRc expression on rickettsial spp.. The housekeeping gene hrtA (17 kDa surface antigen) was used as a control. The negative control for the cDNA synthesis lacking reverse transcriptase is identified by (RTase -). Rickettsial species are identified on the top and the gene names are shown on the left side of the agarose gel. (B) A whole cell lysate from R. rickettsii (1) and insoluble (2) and soluble (3) fractions from R. conorii extracts were isolated and then subjected to Western Blot analysis with anti-APRc antibody. A specific band with approximately 21 kDa was detected. (C) Whole cell lysates (WCL), inner (IM) and outer membrane (OM) fractions from sarkosyl treatment of R. rickettsii and R. conorii extracts were isolated and then subjected to Western Blot analysis with anti-APRc and anti-rOmpB antibody. APRc shares the same localization of rOmpB, an internal marker for outer membrane of Rickettsia spp. Molecular mass markers in kilodaltons (kDa) are shown on the left. (D) For flow cytometric analysis of APRc expression in R. conorii , fixed bacteria were queried for deposition of anti-APRc (orange trace), negative control lacking primay antibody (blue trace), or the positive control anti-OmpB (red trace), a known rickettsial surface protein. After incubation with fluorescent secondary antibody, both anti-APRc and anti-OmpB detected on the bacterial surface (increased fluorescence), indicating accessibility of these target proteins to exogenously applied antibody.
Figure Legend Snippet: RC1339/ APRc is expressed in Rickettsia conorii and Rickettsia rickettsii and accumulates at the outer membrane in both species. (A) RT-PCR analysis of RC1339/ APRc expression on rickettsial spp.. The housekeeping gene hrtA (17 kDa surface antigen) was used as a control. The negative control for the cDNA synthesis lacking reverse transcriptase is identified by (RTase -). Rickettsial species are identified on the top and the gene names are shown on the left side of the agarose gel. (B) A whole cell lysate from R. rickettsii (1) and insoluble (2) and soluble (3) fractions from R. conorii extracts were isolated and then subjected to Western Blot analysis with anti-APRc antibody. A specific band with approximately 21 kDa was detected. (C) Whole cell lysates (WCL), inner (IM) and outer membrane (OM) fractions from sarkosyl treatment of R. rickettsii and R. conorii extracts were isolated and then subjected to Western Blot analysis with anti-APRc and anti-rOmpB antibody. APRc shares the same localization of rOmpB, an internal marker for outer membrane of Rickettsia spp. Molecular mass markers in kilodaltons (kDa) are shown on the left. (D) For flow cytometric analysis of APRc expression in R. conorii , fixed bacteria were queried for deposition of anti-APRc (orange trace), negative control lacking primay antibody (blue trace), or the positive control anti-OmpB (red trace), a known rickettsial surface protein. After incubation with fluorescent secondary antibody, both anti-APRc and anti-OmpB detected on the bacterial surface (increased fluorescence), indicating accessibility of these target proteins to exogenously applied antibody.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Agarose Gel Electrophoresis, Isolation, Western Blot, Marker, Flow Cytometry, Positive Control, Incubation, Fluorescence

2) Product Images from "Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent"

Article Title: Characterization of Microbial Communities and Composition in Constructed Dairy Wetland Wastewater Effluent

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.69.9.5060-5069.2003

DGGE analysis of 16S rRNA fragments of total bacterial population from fecal and manure samples. (A) Gel image from samples collected from manure in February (2mns), calf feces in April (4calfF), cow feces in April (4cowF), calf feces in July (7calfF), and cow feces in July (7cowF). (B) Gel image from samples collected from raw pond in February (2r), collected in December from wetland effluent (we), wetland influent (wi), facultative pond (p), and raw pond (r). Manure (9m1-9m3) and fecal samples were also collected in September from cow (9 cow1-3) and from calf (9 calf 1-3). (C) Numbers before letters represent months that samples were taken; W1 and W2, wetlands 1 and 2; I, influent; E, effluent. P and R represent facultative pond and raw pond, respectively. (D) Numbers before letters represent months that samples were taken. Amplified products were separated on a gradient gel of 30 to 70% denaturant. All labeled bands were excised from the gel, reamplified, and subjected to sequence analysis. These reamplification products were cloned and screened as described in the text.
Figure Legend Snippet: DGGE analysis of 16S rRNA fragments of total bacterial population from fecal and manure samples. (A) Gel image from samples collected from manure in February (2mns), calf feces in April (4calfF), cow feces in April (4cowF), calf feces in July (7calfF), and cow feces in July (7cowF). (B) Gel image from samples collected from raw pond in February (2r), collected in December from wetland effluent (we), wetland influent (wi), facultative pond (p), and raw pond (r). Manure (9m1-9m3) and fecal samples were also collected in September from cow (9 cow1-3) and from calf (9 calf 1-3). (C) Numbers before letters represent months that samples were taken; W1 and W2, wetlands 1 and 2; I, influent; E, effluent. P and R represent facultative pond and raw pond, respectively. (D) Numbers before letters represent months that samples were taken. Amplified products were separated on a gradient gel of 30 to 70% denaturant. All labeled bands were excised from the gel, reamplified, and subjected to sequence analysis. These reamplification products were cloned and screened as described in the text.

Techniques Used: Denaturing Gradient Gel Electrophoresis, Amplification, Labeling, Sequencing, Clone Assay

3) Product Images from "The miR165/166 Mediated Regulatory Module Plays Critical Roles in ABA Homeostasis and Response in Arabidopsis thaliana"

Article Title: The miR165/166 Mediated Regulatory Module Plays Critical Roles in ABA Homeostasis and Response in Arabidopsis thaliana

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1006416

PHB directly upregulates the expression of BG1 . (A) BG1 expression was analyzed using qRT-PCR in PHB : PHB G202G-YFP lines. (B) Analysis of BG1 promoter. The diagram shows a 4.0kb fragment upstream of the first ATG codon. (C) ChIP-qPCR was performed using specific primers corresponding to different promoter regions. (D) EMSA assay showed that PHB binds to the BG1 promoter region. Labeled probes of the BG1 promoter region were incubated with GST-PHB fusion protein. For the competition test, a non-labeled probe was added at 10-fold and 100-fold concentrations.
Figure Legend Snippet: PHB directly upregulates the expression of BG1 . (A) BG1 expression was analyzed using qRT-PCR in PHB : PHB G202G-YFP lines. (B) Analysis of BG1 promoter. The diagram shows a 4.0kb fragment upstream of the first ATG codon. (C) ChIP-qPCR was performed using specific primers corresponding to different promoter regions. (D) EMSA assay showed that PHB binds to the BG1 promoter region. Labeled probes of the BG1 promoter region were incubated with GST-PHB fusion protein. For the competition test, a non-labeled probe was added at 10-fold and 100-fold concentrations.

Techniques Used: Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Labeling, Incubation

PHB promotes ABI4 expression by directly binding to its promoter. (A) ABI4 expression was analyzed in wild type and STTM165/166 2-day-old seedlings using qRT-PCR. (B) ABI4 expression was analyzed in PHB : PHB G202G-YFP lines using qRT-PCR. (C) Analysis of ABI4 promoter. A 3.0kb fragment upstream of ATG was chosen for the promoter analysis. (D) EMSA assay showed that PHB binds to an ABI4 promoter region. Labeled probe of the ABI4 promoter region was incubated with GST-PHB fusion protein. For the competition test, a non-labeled probe was added at 10-fold and 100-fold concentrations.
Figure Legend Snippet: PHB promotes ABI4 expression by directly binding to its promoter. (A) ABI4 expression was analyzed in wild type and STTM165/166 2-day-old seedlings using qRT-PCR. (B) ABI4 expression was analyzed in PHB : PHB G202G-YFP lines using qRT-PCR. (C) Analysis of ABI4 promoter. A 3.0kb fragment upstream of ATG was chosen for the promoter analysis. (D) EMSA assay showed that PHB binds to an ABI4 promoter region. Labeled probe of the ABI4 promoter region was incubated with GST-PHB fusion protein. For the competition test, a non-labeled probe was added at 10-fold and 100-fold concentrations.

Techniques Used: Expressing, Binding Assay, Quantitative RT-PCR, Labeling, Incubation

4) Product Images from "Evidence that a Linear Megaplasmid Encodes Enzymes of Aliphatic Alkene and Epoxide Metabolism and Coenzyme M (2-Mercaptoethanesulfonate) Biosynthesis in Xanthobacter Strain Py2"

Article Title: Evidence that a Linear Megaplasmid Encodes Enzymes of Aliphatic Alkene and Epoxide Metabolism and Coenzyme M (2-Mercaptoethanesulfonate) Biosynthesis in Xanthobacter Strain Py2

Journal: Journal of Bacteriology

doi: 10.1128/JB.183.7.2172-2177.2001

PCR amplification of xamoA (A), xecC (B), and cfxL (C). Lanes 1, total high-molecular-weight DNA from wild-type strain Py2; lanes 2, CHEFE-purified genomic DNA from wild-type strain Py2; lanes 3, CHEFE-purified linear megaplasmid pEK1; lanes 4, total high-molecular-weight DNA from propylene-negative strain Py2.101; lanes 5, CHEFE-purified genomic DNA from propylene-negative strain Py2.101. The positions of the amplified products, which were verified by total sequence analysis, are indicated by the arrows.
Figure Legend Snippet: PCR amplification of xamoA (A), xecC (B), and cfxL (C). Lanes 1, total high-molecular-weight DNA from wild-type strain Py2; lanes 2, CHEFE-purified genomic DNA from wild-type strain Py2; lanes 3, CHEFE-purified linear megaplasmid pEK1; lanes 4, total high-molecular-weight DNA from propylene-negative strain Py2.101; lanes 5, CHEFE-purified genomic DNA from propylene-negative strain Py2.101. The positions of the amplified products, which were verified by total sequence analysis, are indicated by the arrows.

Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Purification, Sequencing

5) Product Images from "Cell Type-Specific Replication of Simian Virus 40 Conferred by Hormone Response Elements in the Late Promoter"

Article Title: Cell Type-Specific Replication of Simian Virus 40 Conferred by Hormone Response Elements in the Late Promoter

Journal: Journal of Virology

doi: 10.1128/JVI.76.13.6762-6770.2002

). (B) Sequences of the wild-type and mutant double-stranded HRE-containing oligonucleotides used in this study. Only the strand corresponding to the viral late RNA is shown. The bases altered in the mutants are underlined and in small capitals. The numbers in parentheses indicate the SV40 nucleotides to which the oligonucleotides correspond in the SV numbering system.
Figure Legend Snippet: ). (B) Sequences of the wild-type and mutant double-stranded HRE-containing oligonucleotides used in this study. Only the strand corresponding to the viral late RNA is shown. The bases altered in the mutants are underlined and in small capitals. The numbers in parentheses indicate the SV40 nucleotides to which the oligonucleotides correspond in the SV numbering system.

Techniques Used: Mutagenesis

6) Product Images from "A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa"

Article Title: A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

Journal: Annals of Clinical Microbiology and Antimicrobials

doi: 10.1186/1476-0711-9-18

PCR detection of oprL and the AES-I HW2 locus from CF sputum . Sputum from 2 patients that had been shown by PFGE screening to contain either AES-I or a unique strain of P. aeruginosa (UCF) was swabbed onto Whatman FTA ® Elute cards. The oprL and HW2 loci were amplified by PCR from these sputum samples (UCF-FTA, AES-I-FTA) or chromosomal DNA (UCF-DNA, AES-I-DNA) purified from the P. aeruginosa strains isolated from the same sputum sample. These strains were also confirmed by PFGE to be AES-I or a unique strain of P. aeruginosa (data not shown).
Figure Legend Snippet: PCR detection of oprL and the AES-I HW2 locus from CF sputum . Sputum from 2 patients that had been shown by PFGE screening to contain either AES-I or a unique strain of P. aeruginosa (UCF) was swabbed onto Whatman FTA ® Elute cards. The oprL and HW2 loci were amplified by PCR from these sputum samples (UCF-FTA, AES-I-FTA) or chromosomal DNA (UCF-DNA, AES-I-DNA) purified from the P. aeruginosa strains isolated from the same sputum sample. These strains were also confirmed by PFGE to be AES-I or a unique strain of P. aeruginosa (data not shown).

Techniques Used: Polymerase Chain Reaction, Amplification, Purification, Isolation

7) Product Images from "G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity"

Article Title: G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

Journal: Scientific Reports

doi: 10.1038/srep43480

Refinement of mAb G196 epitope. ( a ) and ( b ) Western blot analysis using mAb G196 (upper panel) and Coomassie Brilliant blue staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines). ( c ) A representative permutation ELISA analysis of the G196 epitope. N-terminal biotin coupled 11-aa peptides encompassing the G196 epitope SGSGS DLVPR G were permutated at single positions (underlined) to the 19 remaining coded amino acids. The peptides were immobilized onto streptavidin-coated plates, incubated with mAb G196, washed, and then the bound antibody was detected using an HRP-labeled anti-mouse antibody. ( d ) Densitometric analysis of the relative intensities of duplicate-mean spots from the permutation analysis.
Figure Legend Snippet: Refinement of mAb G196 epitope. ( a ) and ( b ) Western blot analysis using mAb G196 (upper panel) and Coomassie Brilliant blue staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines). ( c ) A representative permutation ELISA analysis of the G196 epitope. N-terminal biotin coupled 11-aa peptides encompassing the G196 epitope SGSGS DLVPR G were permutated at single positions (underlined) to the 19 remaining coded amino acids. The peptides were immobilized onto streptavidin-coated plates, incubated with mAb G196, washed, and then the bound antibody was detected using an HRP-labeled anti-mouse antibody. ( d ) Densitometric analysis of the relative intensities of duplicate-mean spots from the permutation analysis.

Techniques Used: Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Incubation, Labeling

Epitope mapping of mAb G196. ( a ) Western blot (WB) analysis using mAb G196 (upper panel) and amide black staining (middle panel) of the purified bacterial proteins. Amino acid sequence alignment of the C-terminal extension of the proteins encoded by pGEX vectors (lower panel): 6P-1, the protein encoded by pGEX-6P-1; 4T-2, pGEX-4T-2; 2 T, pGEX-2T; 3X, pGEX-3X; core, GST(2–212). Bam HI restriction site of the pGEX-2T- or pGEX-4T-2-encoded Gly-Ser (GS, indicated with underlines). ( b ) and ( c ) Western blot analysis using G196 mAb (upper panel) and Coomassie Brilliant blue (CBB) staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines).
Figure Legend Snippet: Epitope mapping of mAb G196. ( a ) Western blot (WB) analysis using mAb G196 (upper panel) and amide black staining (middle panel) of the purified bacterial proteins. Amino acid sequence alignment of the C-terminal extension of the proteins encoded by pGEX vectors (lower panel): 6P-1, the protein encoded by pGEX-6P-1; 4T-2, pGEX-4T-2; 2 T, pGEX-2T; 3X, pGEX-3X; core, GST(2–212). Bam HI restriction site of the pGEX-2T- or pGEX-4T-2-encoded Gly-Ser (GS, indicated with underlines). ( b ) and ( c ) Western blot analysis using G196 mAb (upper panel) and Coomassie Brilliant blue (CBB) staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines).

Techniques Used: Western Blot, Staining, Purification, Sequencing

8) Product Images from "G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity"

Article Title: G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

Journal: Scientific Reports

doi: 10.1038/srep43480

Epitope mapping of mAb G196. ( a ) Western blot (WB) analysis using mAb G196 (upper panel) and amide black staining (middle panel) of the purified bacterial proteins. Amino acid sequence alignment of the C-terminal extension of the proteins encoded by pGEX vectors (lower panel): 6P-1, the protein encoded by pGEX-6P-1; 4T-2, pGEX-4T-2; 2 T, pGEX-2T; 3X, pGEX-3X; core, GST(2–212). Bam HI restriction site of the pGEX-2T- or pGEX-4T-2-encoded Gly-Ser (GS, indicated with underlines). ( b ) and ( c ) Western blot analysis using G196 mAb (upper panel) and Coomassie Brilliant blue (CBB) staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines).
Figure Legend Snippet: Epitope mapping of mAb G196. ( a ) Western blot (WB) analysis using mAb G196 (upper panel) and amide black staining (middle panel) of the purified bacterial proteins. Amino acid sequence alignment of the C-terminal extension of the proteins encoded by pGEX vectors (lower panel): 6P-1, the protein encoded by pGEX-6P-1; 4T-2, pGEX-4T-2; 2 T, pGEX-2T; 3X, pGEX-3X; core, GST(2–212). Bam HI restriction site of the pGEX-2T- or pGEX-4T-2-encoded Gly-Ser (GS, indicated with underlines). ( b ) and ( c ) Western blot analysis using G196 mAb (upper panel) and Coomassie Brilliant blue (CBB) staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines).

Techniques Used: Western Blot, Staining, Purification, Sequencing

9) Product Images from "Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface *"

Article Title: Activation of AMP-activated Protein Kinase Regulates Hippocampal Neuronal pH by Recruiting Na+/H+ Exchanger NHE5 to the Cell Surface *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.555284

The cytoplasmic C terminus of NHE5 binds to the AMPKα2 catalytic subunit in vitro . A , schematic representation of the predicted transmembrane organization of NHE5. B , purified GST and GST-NHE5 fusion proteins encoding peptide segments that span
Figure Legend Snippet: The cytoplasmic C terminus of NHE5 binds to the AMPKα2 catalytic subunit in vitro . A , schematic representation of the predicted transmembrane organization of NHE5. B , purified GST and GST-NHE5 fusion proteins encoding peptide segments that span

Techniques Used: In Vitro, Purification

NHE5 is phosphorylated in vitro by AMPK. A , GST-NHE5 fusion proteins were incubated in vitro with [γ- 32 P]ATP and purified, partially activated, rat liver AMPK in the presence or absence of AMP (0.3 m m ), subjected to SDS-PAGE, and analyzed by autoradiography.
Figure Legend Snippet: NHE5 is phosphorylated in vitro by AMPK. A , GST-NHE5 fusion proteins were incubated in vitro with [γ- 32 P]ATP and purified, partially activated, rat liver AMPK in the presence or absence of AMP (0.3 m m ), subjected to SDS-PAGE, and analyzed by autoradiography.

Techniques Used: In Vitro, Incubation, Purification, SDS Page, Autoradiography

10) Product Images from "Neuroprotection and Remyelination after Autoimmune Demyelination in Mice that Inducibly Overexpress CXCL1"

Article Title: Neuroprotection and Remyelination after Autoimmune Demyelination in Mice that Inducibly Overexpress CXCL1

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2009.080350

Generation of double-Tg mice. Schematic representation of constructs in the mouse strains used to create the GFAP-rtTA × β-Gal-TRE-CXCL1 double-Tg mouse model. Strain 1 (activator), had the reverse-tetracycline transactivator fused to the VP16 viral gene (rtTA-VP16) under the control of the GFAP promoter, and strain 2 (reporter), had the CXCL1 and β-gal genes controlled by the tetracycline response element fused to a minimal CMV promoter (TRE/CMV) ( top ). Performing a F1 cross between these two strains generated animals that expressed rtTA-VP16 in GFAP-positive cells. This in turn, and only in the presence of tetracycline or its derivative doxycycline, drove the expression of the CXCL1 gene product in astrocytes. Representative PCR genotyping of littermates from animals set up to generate double-Tg mice is shown at the bottom ( lanes 1 to 33 ). Lanes containing the 500-bp DNA fragment indicate the presence of GFAP transgene, and the 190-bp band represents CXCL1 gene amplification. Double-Tg animals display both bands, and lanes with no bands indicate animals carrying neither of the transgenes.
Figure Legend Snippet: Generation of double-Tg mice. Schematic representation of constructs in the mouse strains used to create the GFAP-rtTA × β-Gal-TRE-CXCL1 double-Tg mouse model. Strain 1 (activator), had the reverse-tetracycline transactivator fused to the VP16 viral gene (rtTA-VP16) under the control of the GFAP promoter, and strain 2 (reporter), had the CXCL1 and β-gal genes controlled by the tetracycline response element fused to a minimal CMV promoter (TRE/CMV) ( top ). Performing a F1 cross between these two strains generated animals that expressed rtTA-VP16 in GFAP-positive cells. This in turn, and only in the presence of tetracycline or its derivative doxycycline, drove the expression of the CXCL1 gene product in astrocytes. Representative PCR genotyping of littermates from animals set up to generate double-Tg mice is shown at the bottom ( lanes 1 to 33 ). Lanes containing the 500-bp DNA fragment indicate the presence of GFAP transgene, and the 190-bp band represents CXCL1 gene amplification. Double-Tg animals display both bands, and lanes with no bands indicate animals carrying neither of the transgenes.

Techniques Used: Mouse Assay, Construct, Generated, Expressing, Polymerase Chain Reaction, Amplification

11) Product Images from "A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa"

Article Title: A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

Journal: Annals of Clinical Microbiology and Antimicrobials

doi: 10.1186/1476-0711-9-18

PCR detection of oprL and the AES-I HW2 locus from CF sputum . Sputum from 2 patients that had been shown by PFGE screening to contain either AES-I or a unique strain of P. aeruginosa (UCF) was swabbed onto Whatman FTA ® Elute cards. The oprL and HW2 loci were amplified by PCR from these sputum samples (UCF-FTA, AES-I-FTA) or chromosomal DNA (UCF-DNA, AES-I-DNA) purified from the P. aeruginosa strains isolated from the same sputum sample. These strains were also confirmed by PFGE to be AES-I or a unique strain of P. aeruginosa (data not shown).
Figure Legend Snippet: PCR detection of oprL and the AES-I HW2 locus from CF sputum . Sputum from 2 patients that had been shown by PFGE screening to contain either AES-I or a unique strain of P. aeruginosa (UCF) was swabbed onto Whatman FTA ® Elute cards. The oprL and HW2 loci were amplified by PCR from these sputum samples (UCF-FTA, AES-I-FTA) or chromosomal DNA (UCF-DNA, AES-I-DNA) purified from the P. aeruginosa strains isolated from the same sputum sample. These strains were also confirmed by PFGE to be AES-I or a unique strain of P. aeruginosa (data not shown).

Techniques Used: Polymerase Chain Reaction, Amplification, Purification, Isolation

12) Product Images from "Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR"

Article Title: Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.1.245-253.2002

Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.
Figure Legend Snippet: Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

13) Product Images from "Identification of a novel rat hepatic gene induced early by insulin, independently of glucose"

Article Title: Identification of a novel rat hepatic gene induced early by insulin, independently of glucose

Journal: Biochemical Journal

doi: 10.1042/BJ20040586

Tissue distribution analysis of EIIH and Glut2 mRNAs by RT-PCR RT-PCR products (10 μl) were analysed by electrophoresis on a 1.5% (w/v) agarose gel. ( A ) Whole-organ tissue distribution. Li, liver; Pa, pancreas; AT, adipose tissue; SM, skeletal muscle; K, kidney; S, spleen; D, duodenum; I, ileum; B, brain; Pl, placenta; Lu, lung; Te, testis; -, no cDNA. ( B ) Expression in whole pancreas (Pa) and isolated pancreatic islets of Langerhans (IsL). ( C ) Expression in whole brain (B), dissected hypothalamus (H) and pituitary gland (Pi). The sizes of the RT-PCR products were 2489 bp for EIIH, 717 bp for Glut2, 738 bp for GK and 568 bp for cyclophilin. Cyclophilin was used as an indication of cDNA input in the reactions.
Figure Legend Snippet: Tissue distribution analysis of EIIH and Glut2 mRNAs by RT-PCR RT-PCR products (10 μl) were analysed by electrophoresis on a 1.5% (w/v) agarose gel. ( A ) Whole-organ tissue distribution. Li, liver; Pa, pancreas; AT, adipose tissue; SM, skeletal muscle; K, kidney; S, spleen; D, duodenum; I, ileum; B, brain; Pl, placenta; Lu, lung; Te, testis; -, no cDNA. ( B ) Expression in whole pancreas (Pa) and isolated pancreatic islets of Langerhans (IsL). ( C ) Expression in whole brain (B), dissected hypothalamus (H) and pituitary gland (Pi). The sizes of the RT-PCR products were 2489 bp for EIIH, 717 bp for Glut2, 738 bp for GK and 568 bp for cyclophilin. Cyclophilin was used as an indication of cDNA input in the reactions.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Electrophoresis, Agarose Gel Electrophoresis, Expressing, Isolation

Identification of EIIH by DD/RT-PCR and analysis of its induction pattern by insulin ( A ) RNA blot analysis of GK mRNA level of 12-day-old rat hepatocytes in primary cultures stimulated by 100 nM insulin (left-hand panel) and in the liver of pups of the same age after glucose force-feeding (5 g/kg of body mass; right-hand panel) for the indicated times. Total RNA (10 μg) was hybridized with a 1.8 kb GK probe and autoradiographed for 20 h. Lower panels show ethidium-bromide-stained 18 S RNAs used as a control for RNA loading. ( B ) DD/RT-PCR cDNA pattern obtained with primers P6 and T11. The arrow points to EIIH cDNA. ( C ) RNA blot analysis of the induction pattern of EIIH mRNA by insulin. Hybridization of the same membrane as in ( A ) with the 600 bp EIIH probe. The duration of insulin treatment is indicated above each panel.
Figure Legend Snippet: Identification of EIIH by DD/RT-PCR and analysis of its induction pattern by insulin ( A ) RNA blot analysis of GK mRNA level of 12-day-old rat hepatocytes in primary cultures stimulated by 100 nM insulin (left-hand panel) and in the liver of pups of the same age after glucose force-feeding (5 g/kg of body mass; right-hand panel) for the indicated times. Total RNA (10 μg) was hybridized with a 1.8 kb GK probe and autoradiographed for 20 h. Lower panels show ethidium-bromide-stained 18 S RNAs used as a control for RNA loading. ( B ) DD/RT-PCR cDNA pattern obtained with primers P6 and T11. The arrow points to EIIH cDNA. ( C ) RNA blot analysis of the induction pattern of EIIH mRNA by insulin. Hybridization of the same membrane as in ( A ) with the 600 bp EIIH probe. The duration of insulin treatment is indicated above each panel.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern blot, Staining, Hybridization

14) Product Images from "Dual Requirement for the EcR/USP Nuclear Receptor and the dGATAb Factor in an Ecdysone Response in Drosophila melanogaster"

Article Title: Dual Requirement for the EcR/USP Nuclear Receptor and the dGATAb Factor in an Ecdysone Response in Drosophila melanogaster

Journal: Molecular and Cellular Biology

doi:

Expression of GATAb in third-instar larvae. (A) RT-PCR analysis. Total expression of mRNAs for GATAb, Fbp1 , and the ribosomal protein L17A was analyzed at specific developmental stages by quantitative RT-PCR using specific primers. (B) Distribution of GATAb in late-third-instar tissues. Tissues were dissected from late-third-instar larvae and stained with the #Srp anti-GATAb antibody. Nuclear staining was detected in the gut (g), lymph glands (lg), pericardial cells (pc), gonads (go), and fat body (fb). No staining was detected in the other tissues (not shown). (C) Western blot analysis. GATAb-specific bands were identified with the #Srp antibody in a Western blot analysis by comparing the profiles obtained with fat bodies from w1118 or UAS- srp/hs -GAL4 larvae at 25°C with those obtained with fat bodies from UAS- srp/hs -GAL4 larvae after a 1-h heat shock at 37°C (left). On the right is the temporal profile of GATAb protein expression in isolated fat bodies as detected by Western blotting. The protein quantity loaded in each lane was estimated by detection of myosin. The values on the left are molecular sizes in kilodaltons.
Figure Legend Snippet: Expression of GATAb in third-instar larvae. (A) RT-PCR analysis. Total expression of mRNAs for GATAb, Fbp1 , and the ribosomal protein L17A was analyzed at specific developmental stages by quantitative RT-PCR using specific primers. (B) Distribution of GATAb in late-third-instar tissues. Tissues were dissected from late-third-instar larvae and stained with the #Srp anti-GATAb antibody. Nuclear staining was detected in the gut (g), lymph glands (lg), pericardial cells (pc), gonads (go), and fat body (fb). No staining was detected in the other tissues (not shown). (C) Western blot analysis. GATAb-specific bands were identified with the #Srp antibody in a Western blot analysis by comparing the profiles obtained with fat bodies from w1118 or UAS- srp/hs -GAL4 larvae at 25°C with those obtained with fat bodies from UAS- srp/hs -GAL4 larvae after a 1-h heat shock at 37°C (left). On the right is the temporal profile of GATAb protein expression in isolated fat bodies as detected by Western blotting. The protein quantity loaded in each lane was estimated by detection of myosin. The values on the left are molecular sizes in kilodaltons.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Western Blot, Isolation

15) Product Images from "Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions"

Article Title: Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions

Journal: Virology

doi: 10.1016/j.virol.2013.11.008

Requirement of DDX3 for JEV replication. (A) Western blotting analysis of cellular lysate using anti-DDX3 or anti-β-actin antibody after DDX3 knockdown by DDX3 shRNA. (B) Viral titers determined by plaque formation assay after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, Viral titers determined by plaque formation assay at 48 hpi. (C) The JEV genomic RNA levels in JEV infected BHK-21 cells monitored by Q-PCR after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, JEV RNA copy number was determined by Q-PCR at 48 hpi. (D) Western blot analysis on the JEV envelope (E protein) and NS5 protein expression levels in JEV infected BHK-21 cells after DDX3 knockdown. (E) BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h. The amount of virus released into the medium was determined by plaque formation assay at different time points. (F) BHK-21 cells transfected with different amounts of DDX3 shRNA plasmid were infected with JEV (MOI=0.01), 48 h later, the amount of virus released into the medium was determined by plaque formation assay. The differences between means were considered significant at * p
Figure Legend Snippet: Requirement of DDX3 for JEV replication. (A) Western blotting analysis of cellular lysate using anti-DDX3 or anti-β-actin antibody after DDX3 knockdown by DDX3 shRNA. (B) Viral titers determined by plaque formation assay after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, Viral titers determined by plaque formation assay at 48 hpi. (C) The JEV genomic RNA levels in JEV infected BHK-21 cells monitored by Q-PCR after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, JEV RNA copy number was determined by Q-PCR at 48 hpi. (D) Western blot analysis on the JEV envelope (E protein) and NS5 protein expression levels in JEV infected BHK-21 cells after DDX3 knockdown. (E) BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h. The amount of virus released into the medium was determined by plaque formation assay at different time points. (F) BHK-21 cells transfected with different amounts of DDX3 shRNA plasmid were infected with JEV (MOI=0.01), 48 h later, the amount of virus released into the medium was determined by plaque formation assay. The differences between means were considered significant at * p

Techniques Used: Western Blot, shRNA, Plaque Formation Assay, Transfection, Infection, Polymerase Chain Reaction, Expressing, Plasmid Preparation

The effect of exogenous DDX3 and helicase-dead DDX3s on JEV replication. (A) BHK-21 cells were first transfected with DDX3 shRNA plasmid (DDX3i). 24 h later, the cells were transfected with either pcDNA3.1-DDX3-HA, pcDNA3.1-DDX3r-K230E-HA, pcDNA3.1-DDX3r-S382L-HA or pcDNA3.1 plasmid. The viral titers were determined by plaque formation assay at 48 hpi (MOI=0.01). (B) The levels of JEV genome RNA were detected by real-time PCR in JEV-infected cells after the cells were first transfected with the DDX3i plasmid before pcDNA3.1-DDX3r-HA, pcDNA3.1-DDX3r-K230E-HA or pcDNA3.1-DDX3r-S382L-HA plasmid transfection, the control shRNA and pcDNA3.1 plasmid were used as the control. (C) The expression of DDX3 mutant plasmids and the endogenous DDX3 were detected by Western blot using anti-HA monoclonal antibody and anti-DDX3 polyclonal antibody. The β-actin was used as the loading control. (D) The cell viability of BHK-21 cells transfected with either DDX3-shRNA, control-shRNA, DDX3-shRNA/pcDNA3.1-DDX3r, DDX3-shRNA/pcDNA3.1-DDX3r-K230E-HA, DDX3-shRNA/pcDNA3.1-DDX3r-S382L –HA, DDX3-shRNA /pcDNA3.1 or control shRNA/pcDNA3.1 was detected by CytoTox 96 ® NonRadioactive Cytotoxicity Assay (Promega) at 72 h after transfection. The differences between means were considered significant at * p
Figure Legend Snippet: The effect of exogenous DDX3 and helicase-dead DDX3s on JEV replication. (A) BHK-21 cells were first transfected with DDX3 shRNA plasmid (DDX3i). 24 h later, the cells were transfected with either pcDNA3.1-DDX3-HA, pcDNA3.1-DDX3r-K230E-HA, pcDNA3.1-DDX3r-S382L-HA or pcDNA3.1 plasmid. The viral titers were determined by plaque formation assay at 48 hpi (MOI=0.01). (B) The levels of JEV genome RNA were detected by real-time PCR in JEV-infected cells after the cells were first transfected with the DDX3i plasmid before pcDNA3.1-DDX3r-HA, pcDNA3.1-DDX3r-K230E-HA or pcDNA3.1-DDX3r-S382L-HA plasmid transfection, the control shRNA and pcDNA3.1 plasmid were used as the control. (C) The expression of DDX3 mutant plasmids and the endogenous DDX3 were detected by Western blot using anti-HA monoclonal antibody and anti-DDX3 polyclonal antibody. The β-actin was used as the loading control. (D) The cell viability of BHK-21 cells transfected with either DDX3-shRNA, control-shRNA, DDX3-shRNA/pcDNA3.1-DDX3r, DDX3-shRNA/pcDNA3.1-DDX3r-K230E-HA, DDX3-shRNA/pcDNA3.1-DDX3r-S382L –HA, DDX3-shRNA /pcDNA3.1 or control shRNA/pcDNA3.1 was detected by CytoTox 96 ® NonRadioactive Cytotoxicity Assay (Promega) at 72 h after transfection. The differences between means were considered significant at * p

Techniques Used: Transfection, shRNA, Plasmid Preparation, Plaque Formation Assay, Real-time Polymerase Chain Reaction, Infection, Expressing, Mutagenesis, Western Blot, Cytotoxicity Assay

16) Product Images from "Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR"

Article Title: Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.1.245-253.2002

Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.
Figure Legend Snippet: Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

17) Product Images from "G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity"

Article Title: G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

Journal: Scientific Reports

doi: 10.1038/srep43480

Epitope mapping of mAb G196. ( a ) Western blot (WB) analysis using mAb G196 (upper panel) and amide black staining (middle panel) of the purified bacterial proteins. Amino acid sequence alignment of the C-terminal extension of the proteins encoded by pGEX vectors (lower panel): 6P-1, the protein encoded by pGEX-6P-1; 4T-2, pGEX-4T-2; 2 T, pGEX-2T; 3X, pGEX-3X; core, GST(2–212). Bam HI restriction site of the pGEX-2T- or pGEX-4T-2-encoded Gly-Ser (GS, indicated with underlines). ( b ) and ( c ) Western blot analysis using G196 mAb (upper panel) and Coomassie Brilliant blue (CBB) staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines).
Figure Legend Snippet: Epitope mapping of mAb G196. ( a ) Western blot (WB) analysis using mAb G196 (upper panel) and amide black staining (middle panel) of the purified bacterial proteins. Amino acid sequence alignment of the C-terminal extension of the proteins encoded by pGEX vectors (lower panel): 6P-1, the protein encoded by pGEX-6P-1; 4T-2, pGEX-4T-2; 2 T, pGEX-2T; 3X, pGEX-3X; core, GST(2–212). Bam HI restriction site of the pGEX-2T- or pGEX-4T-2-encoded Gly-Ser (GS, indicated with underlines). ( b ) and ( c ) Western blot analysis using G196 mAb (upper panel) and Coomassie Brilliant blue (CBB) staining (middle panel) of the bacterially expressed proteins shown in the lower panel. Bam HI and Eco RI restriction sites of the pGEX6P-1-encoded Gly-Ser (GS) and Glu-Phe (EF), respectively (indicated with underlines).

Techniques Used: Western Blot, Staining, Purification, Sequencing

18) Product Images from "Albidovulum inexpectatum gen. nov., sp. nov., a Nonphotosynthetic and Slightly Thermophilic Bacterium from a Marine Hot Spring That Is Very Closely Related to Members of the Photosynthetic Genus Rhodovulum"

Article Title: Albidovulum inexpectatum gen. nov., sp. nov., a Nonphotosynthetic and Slightly Thermophilic Bacterium from a Marine Hot Spring That Is Very Closely Related to Members of the Photosynthetic Genus Rhodovulum

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.9.4266-4273.2002

Agarose gel electrophoresis of PCR-amplified partial puf genes with an annealing temperature of 48°C. Lane 1, marker IV (Roche Molecular Biochemicals). Amplification products were generated with primers PUBM1 and PUBM2 from Rhodovulum sulfidophilum DNA (lane 2), Rhodovulum euryhalinum DNA (lane 4), and FRR-10 T DNA (lane 7), and with PULM1 and PULM2 from Rhodovulum sulfidophilum DNA (lane 3), Rhodovulum euryhalinum DNA (lane 5), Roseococcus thiosulfatophilus DNA (lane 6), and FRR-10 T DNA (lane 8).
Figure Legend Snippet: Agarose gel electrophoresis of PCR-amplified partial puf genes with an annealing temperature of 48°C. Lane 1, marker IV (Roche Molecular Biochemicals). Amplification products were generated with primers PUBM1 and PUBM2 from Rhodovulum sulfidophilum DNA (lane 2), Rhodovulum euryhalinum DNA (lane 4), and FRR-10 T DNA (lane 7), and with PULM1 and PULM2 from Rhodovulum sulfidophilum DNA (lane 3), Rhodovulum euryhalinum DNA (lane 5), Roseococcus thiosulfatophilus DNA (lane 6), and FRR-10 T DNA (lane 8).

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

19) Product Images from "Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA"

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.17.5210-5219.2003

Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a PCR at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate DNA bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.
Figure Legend Snippet: Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a PCR at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate DNA bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.

Techniques Used: Clone Assay, Polymerase Chain Reaction, Generated, Southern Blot, Staining, Agarose Gel Electrophoresis, Sequencing

20) Product Images from "Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions"

Article Title: Cellular DDX3 regulates Japanese encephalitis virus replication by interacting with viral un-translated regions

Journal: Virology

doi: 10.1016/j.virol.2013.11.008

Requirement of DDX3 for JEV replication. (A) Western blotting analysis of cellular lysate using anti-DDX3 or anti-β-actin antibody after DDX3 knockdown by DDX3 shRNA. (B) Viral titers determined by plaque formation assay after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, Viral titers determined by plaque formation assay at 48 hpi. (C) The JEV genomic RNA levels in JEV infected BHK-21 cells monitored by Q-PCR after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, JEV RNA copy number was determined by Q-PCR at 48 hpi. (D) Western blot analysis on the JEV envelope (E protein) and NS5 protein expression levels in JEV infected BHK-21 cells after DDX3 knockdown. (E) BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h. The amount of virus released into the medium was determined by plaque formation assay at different time points. (F) BHK-21 cells transfected with different amounts of DDX3 shRNA plasmid were infected with JEV (MOI=0.01), 48 h later, the amount of virus released into the medium was determined by plaque formation assay. The differences between means were considered significant at * p
Figure Legend Snippet: Requirement of DDX3 for JEV replication. (A) Western blotting analysis of cellular lysate using anti-DDX3 or anti-β-actin antibody after DDX3 knockdown by DDX3 shRNA. (B) Viral titers determined by plaque formation assay after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, Viral titers determined by plaque formation assay at 48 hpi. (C) The JEV genomic RNA levels in JEV infected BHK-21 cells monitored by Q-PCR after DDX3 knockdown. BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h, JEV RNA copy number was determined by Q-PCR at 48 hpi. (D) Western blot analysis on the JEV envelope (E protein) and NS5 protein expression levels in JEV infected BHK-21 cells after DDX3 knockdown. (E) BHK-21 cells transfected with the DDX3 shRNA were infected with JEV (MOI=0.01) for 48 h. The amount of virus released into the medium was determined by plaque formation assay at different time points. (F) BHK-21 cells transfected with different amounts of DDX3 shRNA plasmid were infected with JEV (MOI=0.01), 48 h later, the amount of virus released into the medium was determined by plaque formation assay. The differences between means were considered significant at * p

Techniques Used: Western Blot, shRNA, Plaque Formation Assay, Transfection, Infection, Polymerase Chain Reaction, Expressing, Plasmid Preparation

The effect of exogenous DDX3 and helicase-dead DDX3s on JEV replication. (A) BHK-21 cells were first transfected with DDX3 shRNA plasmid (DDX3i). 24 h later, the cells were transfected with either pcDNA3.1-DDX3-HA, pcDNA3.1-DDX3r-K230E-HA, pcDNA3.1-DDX3r-S382L-HA or pcDNA3.1 plasmid. The viral titers were determined by plaque formation assay at 48 hpi (MOI=0.01). (B) The levels of JEV genome RNA were detected by real-time PCR in JEV-infected cells after the cells were first transfected with the DDX3i plasmid before pcDNA3.1-DDX3r-HA, pcDNA3.1-DDX3r-K230E-HA or pcDNA3.1-DDX3r-S382L-HA plasmid transfection, the control shRNA and pcDNA3.1 plasmid were used as the control. (C) The expression of DDX3 mutant plasmids and the endogenous DDX3 were detected by Western blot using anti-HA monoclonal antibody and anti-DDX3 polyclonal antibody. The β-actin was used as the loading control. (D) The cell viability of BHK-21 cells transfected with either DDX3-shRNA, control-shRNA, DDX3-shRNA/pcDNA3.1-DDX3r, DDX3-shRNA/pcDNA3.1-DDX3r-K230E-HA, DDX3-shRNA/pcDNA3.1-DDX3r-S382L –HA, DDX3-shRNA /pcDNA3.1 or control shRNA/pcDNA3.1 was detected by CytoTox 96 ® NonRadioactive Cytotoxicity Assay (Promega) at 72 h after transfection. The differences between means were considered significant at * p
Figure Legend Snippet: The effect of exogenous DDX3 and helicase-dead DDX3s on JEV replication. (A) BHK-21 cells were first transfected with DDX3 shRNA plasmid (DDX3i). 24 h later, the cells were transfected with either pcDNA3.1-DDX3-HA, pcDNA3.1-DDX3r-K230E-HA, pcDNA3.1-DDX3r-S382L-HA or pcDNA3.1 plasmid. The viral titers were determined by plaque formation assay at 48 hpi (MOI=0.01). (B) The levels of JEV genome RNA were detected by real-time PCR in JEV-infected cells after the cells were first transfected with the DDX3i plasmid before pcDNA3.1-DDX3r-HA, pcDNA3.1-DDX3r-K230E-HA or pcDNA3.1-DDX3r-S382L-HA plasmid transfection, the control shRNA and pcDNA3.1 plasmid were used as the control. (C) The expression of DDX3 mutant plasmids and the endogenous DDX3 were detected by Western blot using anti-HA monoclonal antibody and anti-DDX3 polyclonal antibody. The β-actin was used as the loading control. (D) The cell viability of BHK-21 cells transfected with either DDX3-shRNA, control-shRNA, DDX3-shRNA/pcDNA3.1-DDX3r, DDX3-shRNA/pcDNA3.1-DDX3r-K230E-HA, DDX3-shRNA/pcDNA3.1-DDX3r-S382L –HA, DDX3-shRNA /pcDNA3.1 or control shRNA/pcDNA3.1 was detected by CytoTox 96 ® NonRadioactive Cytotoxicity Assay (Promega) at 72 h after transfection. The differences between means were considered significant at * p

Techniques Used: Transfection, shRNA, Plasmid Preparation, Plaque Formation Assay, Real-time Polymerase Chain Reaction, Infection, Expressing, Mutagenesis, Western Blot, Cytotoxicity Assay

21) Product Images from "Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR"

Article Title: Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.1.245-253.2002

Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.
Figure Legend Snippet: Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

22) Product Images from "Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR"

Article Title: Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.68.1.245-253.2002

Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.
Figure Legend Snippet: Quantification of copies of the amoA gene (A) and the Nitrospira 16S rDNA gene (B). Numbers of determined copies were calculated by using cPCR as described in Materials and Methods with a PCR product of clone M-20 (A) or a plasmid containing the 16S rDNA gene of Nitrospira (B). Error bars indicate the standard deviations of the means ( n = 3 or 4). The dashed lines indicate a ratio of actual number of copies to determined number of copies of 1:1.

Techniques Used: Polymerase Chain Reaction, Plasmid Preparation

23) Product Images from "Host Heterogeneous Ribonucleoprotein K (hnRNP K) as a Potential Target to Suppress Hepatitis B Virus ReplicationTargeting of a Host Protein to Suppress Hepatitis B Virus Replication"

Article Title: Host Heterogeneous Ribonucleoprotein K (hnRNP K) as a Potential Target to Suppress Hepatitis B Virus ReplicationTargeting of a Host Protein to Suppress Hepatitis B Virus Replication

Journal: PLoS Medicine

doi: 10.1371/journal.pmed.0020163

hnRNP K siRNAs Down-Regulate HBV Viral Replication (A) HepG2 cells were co-transfected with 1752A full-length replicative HBV clone either with or without hnRNP K siRNA (2 μg). Non-silencing (Non-T) and lamin A/C (Lamin) siRNAs were used as controls. hnRNP K expression was measured by quantitative real-time RT-PCR. (B) HBV viral load was quantitated by real-time PCR in cells transfected as described in (A). (C) Lamin A/C expression was measured from real-time RT-PCR. Ratios were normalized to 100% for the non-transfected cells. The results represent two independent samples; standard deviations are shown. Black columns represent either non-transfected cells or cells transfected with non-silencing siRNA. White columns represent cells co-transfected with HBV and lamin A/C siRNA. Grey columns represent cells co-transfected with HBV and hnRNP K siRNAs (A, Dharmacon; B, Qiagen; C, Proligo).
Figure Legend Snippet: hnRNP K siRNAs Down-Regulate HBV Viral Replication (A) HepG2 cells were co-transfected with 1752A full-length replicative HBV clone either with or without hnRNP K siRNA (2 μg). Non-silencing (Non-T) and lamin A/C (Lamin) siRNAs were used as controls. hnRNP K expression was measured by quantitative real-time RT-PCR. (B) HBV viral load was quantitated by real-time PCR in cells transfected as described in (A). (C) Lamin A/C expression was measured from real-time RT-PCR. Ratios were normalized to 100% for the non-transfected cells. The results represent two independent samples; standard deviations are shown. Black columns represent either non-transfected cells or cells transfected with non-silencing siRNA. White columns represent cells co-transfected with HBV and lamin A/C siRNA. Grey columns represent cells co-transfected with HBV and hnRNP K siRNAs (A, Dharmacon; B, Qiagen; C, Proligo).

Techniques Used: Transfection, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Evidence for the Involvement of a Host Cellular Protein in Enh II Activity and HBV Replication (A) Electrophoretic mobility shift assays were performed using HepG2 nuclear extracts with four different probes. Probe 1, lanes 1–4; probe 2 (1752A), lanes 5–8; probe 3, lanes 9–12; probe 4 (1752G), lanes 13–16. Each set of probes contains increasing concentrations (0.0 μg, 0.05 μg, 0.10 μg, and 0.15 μg) of non-specific competitor DNA [poly-(dI)-poly-(dC)], respectively. (B) 40 μg of nuclear protein extracts obtained from HepG2 cells was allowed to bind onto 5 mg Dynabeads M-280 streptavidin-biotin-oligonucleotides in the presence of 2:1 (w/w) ratio of non-specific competitor DNA poly (dI–dC). 1-D isoelectric focusing was followed by 2-D vertical separation on SDS-PAGE (10%). The estimated molecular weight of the specific protein spots detected by silver staining (arrow) is indicated.
Figure Legend Snippet: Evidence for the Involvement of a Host Cellular Protein in Enh II Activity and HBV Replication (A) Electrophoretic mobility shift assays were performed using HepG2 nuclear extracts with four different probes. Probe 1, lanes 1–4; probe 2 (1752A), lanes 5–8; probe 3, lanes 9–12; probe 4 (1752G), lanes 13–16. Each set of probes contains increasing concentrations (0.0 μg, 0.05 μg, 0.10 μg, and 0.15 μg) of non-specific competitor DNA [poly-(dI)-poly-(dC)], respectively. (B) 40 μg of nuclear protein extracts obtained from HepG2 cells was allowed to bind onto 5 mg Dynabeads M-280 streptavidin-biotin-oligonucleotides in the presence of 2:1 (w/w) ratio of non-specific competitor DNA poly (dI–dC). 1-D isoelectric focusing was followed by 2-D vertical separation on SDS-PAGE (10%). The estimated molecular weight of the specific protein spots detected by silver staining (arrow) is indicated.

Techniques Used: Activity Assay, Electrophoretic Mobility Shift Assay, SDS Page, Molecular Weight, Silver Staining

Effect of Nucleotide 1752 Base Substitution on Enh II Activity (A) The Enh II is located just upstream of the core promoter. The minimum sequence for enhancer activity has been previously defined at nucleotide 1687–1805 as shown. (B) Site-directed mutagenesis on nucleotide 1752 in the Enh II (1752A, 1752G, 1752T, and 1752C) and amplified fragments were inserted upstream of the SV40 promoter in an enhancerless luciferase reporter vector. (C–F) Four cell lines—HepG2, PLC/PRF/5, SKHep1, and HCCM, all derived from human hepatocellular carcinomas—were transfected with the respective Enh II clones (1752A, 1752G, 1752T, and 1752C). For each cell type, the first column (“+”) represents the luciferase activity of the internal positive control (promoter and enhancer). The second column (“−”) represents the activity of the internal negative control (promoter only). The other columns of each cell line represent the vectors with the promoter and the Enh II (nucleotide 1752A, 1752G, 1752T, or 1752C). Results of the luciferase assay were normalized to the level of the internal positive control (arbitrarily set at 100%).
Figure Legend Snippet: Effect of Nucleotide 1752 Base Substitution on Enh II Activity (A) The Enh II is located just upstream of the core promoter. The minimum sequence for enhancer activity has been previously defined at nucleotide 1687–1805 as shown. (B) Site-directed mutagenesis on nucleotide 1752 in the Enh II (1752A, 1752G, 1752T, and 1752C) and amplified fragments were inserted upstream of the SV40 promoter in an enhancerless luciferase reporter vector. (C–F) Four cell lines—HepG2, PLC/PRF/5, SKHep1, and HCCM, all derived from human hepatocellular carcinomas—were transfected with the respective Enh II clones (1752A, 1752G, 1752T, and 1752C). For each cell type, the first column (“+”) represents the luciferase activity of the internal positive control (promoter and enhancer). The second column (“−”) represents the activity of the internal negative control (promoter only). The other columns of each cell line represent the vectors with the promoter and the Enh II (nucleotide 1752A, 1752G, 1752T, or 1752C). Results of the luciferase assay were normalized to the level of the internal positive control (arbitrarily set at 100%).

Techniques Used: Activity Assay, Sequencing, Mutagenesis, Amplification, Luciferase, Plasmid Preparation, Planar Chromatography, Derivative Assay, Transfection, Clone Assay, Positive Control, Negative Control

hnRNP K Is Involved in Modulating Viral Replication HepG2 cells were co-transfected with full-length replicative HBV clones (indicated by “+”) 1752A, 1752ΔG, 1752ΔT, and 1752ΔC (see Methods and Figure S1 ) with increasing dosages (50, 250, and 1,250 ng/μl) of hnRNP K variant 2 (v2) or variant 3 (v3) as indicated. pcDNA 3.1 serves as a control. Transfections were performed in duplicate; standard deviations are shown.
Figure Legend Snippet: hnRNP K Is Involved in Modulating Viral Replication HepG2 cells were co-transfected with full-length replicative HBV clones (indicated by “+”) 1752A, 1752ΔG, 1752ΔT, and 1752ΔC (see Methods and Figure S1 ) with increasing dosages (50, 250, and 1,250 ng/μl) of hnRNP K variant 2 (v2) or variant 3 (v3) as indicated. pcDNA 3.1 serves as a control. Transfections were performed in duplicate; standard deviations are shown.

Techniques Used: Transfection, Clone Assay, Variant Assay

24) Product Images from "Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA"

Article Title: Identification of a Differentially Expressed Oligopeptide Binding Protein (OppA2) in Streptococcus uberis by Representational Difference Analysis of cDNA

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.17.5210-5219.2003

Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a PCR at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate DNA bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.
Figure Legend Snippet: Cloning the full-length oppA2 gene. (a) Schematic of how 5′ and 3′ regions of the oppA2 gene were cloned. Gene-specific primers SP1 and SP2 (HAP F and HAP R) were used in concert with primers directed against pneumococcal sequences RP1 or RP2 and were used in a PCR at low annealing temperature to encourage mispriming of the pneumococcal primers. (b) Cloning the 3′ oppA2 region. Primer RP2 misprimed close to the oppA2 gene and generated a product that was detected by Southern blotting by using pDub3 insert as a probe (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products). (c) Cloning the 5′ sequence of oppA2 (lane M, GeneRuler 1-kb ladder; center lane, ethidium bromide-stained agarose gel of PCR products; and rightmost lane, Southern blot of PCR products probed with pDub3). Arrowheads indicate DNA bands taken for cloning. Molecular mass of GeneRuler 1-kb ladder (MBI) shown in kilobases.

Techniques Used: Clone Assay, Polymerase Chain Reaction, Generated, Southern Blot, Staining, Agarose Gel Electrophoresis, Sequencing

Related Articles

Clone Assay:

Article Title: Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution
Article Snippet: .. The vector pGEX-6P-1 (GE Healthcare, USA) was used for gene cloning and protein expression. .. Serratia sp. and E. coli were grown in LB medium (tryptone 1 %, yeast extract 0.5 % and NaCl 1 % w/v) at 28 and 37 °C, respectively. p -nitrophenyl esters: p -NP acetate (C2), p -NP butyrate (C4), p -NP hexanoate (C6), p -NP caprylate (C8), p -NP laurate (C12) and p -NP palmitate (C16) were all purchased from Sigma (St. Louis, MO, USA).

Subcloning:

Article Title: Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants
Article Snippet: .. GST-tagged TKTKK1 construction and Western blot hybridization The GST -tagged pGEX-6P-1 vector (GE Healthcare Life Sciences) was used for sub-cloning TKTKK1 by fusing with the GST sequence at its 3′-terminal. .. After verification by sequencing, the new plasmid pGEX-6P-1 with GST-TKTKK1 was transformed into the E. coli BL21.

Purification:

Article Title: Cloning and Characterization of a 2-Cys Peroxiredoxin from Babesiagibsoni
Article Snippet: .. The PCR product was digested with Eco RI andXho I and then ligated to the pGEX-6P1 vector containing an ORF encoding a glutathione S -transferase (GST)-fusion protein (GE Healthcare, Piscataway, NJ, U.S.A.). rBgTPx-1 was expressed as a GST-fusion protein in E. coli and purified using Glutathione-Sepharose 4B beads (GE Healthcare) as previously described [ ]. .. Then, GST was cleaved by PreScission protease (GE Healthcare) according to the manufacturer’s instructions.

Article Title: A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma
Article Snippet: .. Briefly, Rosetta (DE3) Escherichia coli cells were transformed with the pGEX-6P-1-GST vector or pGEX-6P-1-GST-PFKL, and then, expression was induced using 0.5 mM IPTG at 16 °C for 16 h. The E. coli were lysed, and the extracts were incubated with glutathione–Sepharose 4B beads (17075601; GE Healthcare Biosciences AB) at 4 °C for 1 h. The beads were then incubated with purified GFP-tagged A20, which were prepared through IP, for an additional 4 h. Proteins that had interacted were eluted in elution buffer (50 mM Tris-HCl pH 8.0 and 20 mM reduced glutathione) and were subjected to immunoblotting using anti-GFP antibody. .. Extracts from E. coli expressing only a GST tag were used as the negative control.

Sequencing:

Article Title: A JAZ Protein in Astragalus sinicus Interacts with a Leghemoglobin through the TIFY Domain and Is Involved in Nodule Development and Nitrogen Fixation
Article Snippet: .. For GST-AsB2510 fusion proteins expression, the coding sequence of AsB2510 was digested by Bam HI and Xho I and inserted into a pGEX-6p-1 vector (GE Healthcare, Germany), generating the recombinant plasmid pGEX-6p-1-Lb. .. E . coli Rosetta (DE3) (Novagen) harboring pGEX-6p-1-Lb was induced with 1mmol/L IPTG for 12 h at 20°C.

Article Title: Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants
Article Snippet: .. GST-tagged TKTKK1 construction and Western blot hybridization The GST -tagged pGEX-6P-1 vector (GE Healthcare Life Sciences) was used for sub-cloning TKTKK1 by fusing with the GST sequence at its 3′-terminal. .. After verification by sequencing, the new plasmid pGEX-6P-1 with GST-TKTKK1 was transformed into the E. coli BL21.

Incubation:

Article Title: A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma
Article Snippet: .. Briefly, Rosetta (DE3) Escherichia coli cells were transformed with the pGEX-6P-1-GST vector or pGEX-6P-1-GST-PFKL, and then, expression was induced using 0.5 mM IPTG at 16 °C for 16 h. The E. coli were lysed, and the extracts were incubated with glutathione–Sepharose 4B beads (17075601; GE Healthcare Biosciences AB) at 4 °C for 1 h. The beads were then incubated with purified GFP-tagged A20, which were prepared through IP, for an additional 4 h. Proteins that had interacted were eluted in elution buffer (50 mM Tris-HCl pH 8.0 and 20 mM reduced glutathione) and were subjected to immunoblotting using anti-GFP antibody. .. Extracts from E. coli expressing only a GST tag were used as the negative control.

Expressing:

Article Title: A JAZ Protein in Astragalus sinicus Interacts with a Leghemoglobin through the TIFY Domain and Is Involved in Nodule Development and Nitrogen Fixation
Article Snippet: .. For GST-AsB2510 fusion proteins expression, the coding sequence of AsB2510 was digested by Bam HI and Xho I and inserted into a pGEX-6p-1 vector (GE Healthcare, Germany), generating the recombinant plasmid pGEX-6p-1-Lb. .. E . coli Rosetta (DE3) (Novagen) harboring pGEX-6p-1-Lb was induced with 1mmol/L IPTG for 12 h at 20°C.

Article Title: Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution
Article Snippet: .. The vector pGEX-6P-1 (GE Healthcare, USA) was used for gene cloning and protein expression. .. Serratia sp. and E. coli were grown in LB medium (tryptone 1 %, yeast extract 0.5 % and NaCl 1 % w/v) at 28 and 37 °C, respectively. p -nitrophenyl esters: p -NP acetate (C2), p -NP butyrate (C4), p -NP hexanoate (C6), p -NP caprylate (C8), p -NP laurate (C12) and p -NP palmitate (C16) were all purchased from Sigma (St. Louis, MO, USA).

Article Title: EF1A interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication
Article Snippet: .. PCR products were subcloned into a prokaryotic expression pGEX-6p-1 vector (GE Healthcare). .. Recombinant expression plasmid was designated as pGEX-TGEV-N and confirmed by DNA sequencing.

Article Title: Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii
Article Snippet: .. Expression of recombinant TgPuf1 Puf domain in Escherichia coli To express the conserved RNA-binding domain of TgPuf1 in bacteria, PCR was performed with T. gondii cDNA using two primers (CGGGATCC AGAAAAGGCGACTCAAAAG and ATAAGAATGCGGCCGC GTCACTGAAACCTGAGATG) designed to clone at the Bam HI and Not I sites of the expression vector pGEX-6P-1 (GE Healthcare). ..

Article Title: A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma
Article Snippet: .. Briefly, Rosetta (DE3) Escherichia coli cells were transformed with the pGEX-6P-1-GST vector or pGEX-6P-1-GST-PFKL, and then, expression was induced using 0.5 mM IPTG at 16 °C for 16 h. The E. coli were lysed, and the extracts were incubated with glutathione–Sepharose 4B beads (17075601; GE Healthcare Biosciences AB) at 4 °C for 1 h. The beads were then incubated with purified GFP-tagged A20, which were prepared through IP, for an additional 4 h. Proteins that had interacted were eluted in elution buffer (50 mM Tris-HCl pH 8.0 and 20 mM reduced glutathione) and were subjected to immunoblotting using anti-GFP antibody. .. Extracts from E. coli expressing only a GST tag were used as the negative control.

Article Title: A Second Lysine-Specific Serine Protease from Lysobacter sp. Strain IB-9374
Article Snippet: .. The insert DNA was digested with BamHI and EcoRI and then ligated into an expression vector of GST fusion protein, pGEX-6P-1 (Amersham Pharmacia Biotech AB), and digested with the same restriction enzyme, giving pGEX-lepB (Fig. ). ..

Polymerase Chain Reaction:

Article Title: EF1A interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication
Article Snippet: .. PCR products were subcloned into a prokaryotic expression pGEX-6p-1 vector (GE Healthcare). .. Recombinant expression plasmid was designated as pGEX-TGEV-N and confirmed by DNA sequencing.

Article Title: Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii
Article Snippet: .. Expression of recombinant TgPuf1 Puf domain in Escherichia coli To express the conserved RNA-binding domain of TgPuf1 in bacteria, PCR was performed with T. gondii cDNA using two primers (CGGGATCC AGAAAAGGCGACTCAAAAG and ATAAGAATGCGGCCGC GTCACTGAAACCTGAGATG) designed to clone at the Bam HI and Not I sites of the expression vector pGEX-6P-1 (GE Healthcare). ..

Article Title: Cloning and Characterization of a 2-Cys Peroxiredoxin from Babesiagibsoni
Article Snippet: .. The PCR product was digested with Eco RI andXho I and then ligated to the pGEX-6P1 vector containing an ORF encoding a glutathione S -transferase (GST)-fusion protein (GE Healthcare, Piscataway, NJ, U.S.A.). rBgTPx-1 was expressed as a GST-fusion protein in E. coli and purified using Glutathione-Sepharose 4B beads (GE Healthcare) as previously described [ ]. .. Then, GST was cleaved by PreScission protease (GE Healthcare) according to the manufacturer’s instructions.

Western Blot:

Article Title: Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants
Article Snippet: .. GST-tagged TKTKK1 construction and Western blot hybridization The GST -tagged pGEX-6P-1 vector (GE Healthcare Life Sciences) was used for sub-cloning TKTKK1 by fusing with the GST sequence at its 3′-terminal. .. After verification by sequencing, the new plasmid pGEX-6P-1 with GST-TKTKK1 was transformed into the E. coli BL21.

RNA Binding Assay:

Article Title: Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii
Article Snippet: .. Expression of recombinant TgPuf1 Puf domain in Escherichia coli To express the conserved RNA-binding domain of TgPuf1 in bacteria, PCR was performed with T. gondii cDNA using two primers (CGGGATCC AGAAAAGGCGACTCAAAAG and ATAAGAATGCGGCCGC GTCACTGAAACCTGAGATG) designed to clone at the Bam HI and Not I sites of the expression vector pGEX-6P-1 (GE Healthcare). ..

Recombinant:

Article Title: A JAZ Protein in Astragalus sinicus Interacts with a Leghemoglobin through the TIFY Domain and Is Involved in Nodule Development and Nitrogen Fixation
Article Snippet: .. For GST-AsB2510 fusion proteins expression, the coding sequence of AsB2510 was digested by Bam HI and Xho I and inserted into a pGEX-6p-1 vector (GE Healthcare, Germany), generating the recombinant plasmid pGEX-6p-1-Lb. .. E . coli Rosetta (DE3) (Novagen) harboring pGEX-6p-1-Lb was induced with 1mmol/L IPTG for 12 h at 20°C.

Article Title: Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii
Article Snippet: .. Expression of recombinant TgPuf1 Puf domain in Escherichia coli To express the conserved RNA-binding domain of TgPuf1 in bacteria, PCR was performed with T. gondii cDNA using two primers (CGGGATCC AGAAAAGGCGACTCAAAAG and ATAAGAATGCGGCCGC GTCACTGAAACCTGAGATG) designed to clone at the Bam HI and Not I sites of the expression vector pGEX-6P-1 (GE Healthcare). ..

Transformation Assay:

Article Title: A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma
Article Snippet: .. Briefly, Rosetta (DE3) Escherichia coli cells were transformed with the pGEX-6P-1-GST vector or pGEX-6P-1-GST-PFKL, and then, expression was induced using 0.5 mM IPTG at 16 °C for 16 h. The E. coli were lysed, and the extracts were incubated with glutathione–Sepharose 4B beads (17075601; GE Healthcare Biosciences AB) at 4 °C for 1 h. The beads were then incubated with purified GFP-tagged A20, which were prepared through IP, for an additional 4 h. Proteins that had interacted were eluted in elution buffer (50 mM Tris-HCl pH 8.0 and 20 mM reduced glutathione) and were subjected to immunoblotting using anti-GFP antibody. .. Extracts from E. coli expressing only a GST tag were used as the negative control.

Plasmid Preparation:

Article Title: A JAZ Protein in Astragalus sinicus Interacts with a Leghemoglobin through the TIFY Domain and Is Involved in Nodule Development and Nitrogen Fixation
Article Snippet: .. For GST-AsB2510 fusion proteins expression, the coding sequence of AsB2510 was digested by Bam HI and Xho I and inserted into a pGEX-6p-1 vector (GE Healthcare, Germany), generating the recombinant plasmid pGEX-6p-1-Lb. .. E . coli Rosetta (DE3) (Novagen) harboring pGEX-6p-1-Lb was induced with 1mmol/L IPTG for 12 h at 20°C.

Article Title: Characterization of a cold-active esterase from Serratia sp. and improvement of thermostability by directed evolution
Article Snippet: .. The vector pGEX-6P-1 (GE Healthcare, USA) was used for gene cloning and protein expression. .. Serratia sp. and E. coli were grown in LB medium (tryptone 1 %, yeast extract 0.5 % and NaCl 1 % w/v) at 28 and 37 °C, respectively. p -nitrophenyl esters: p -NP acetate (C2), p -NP butyrate (C4), p -NP hexanoate (C6), p -NP caprylate (C8), p -NP laurate (C12) and p -NP palmitate (C16) were all purchased from Sigma (St. Louis, MO, USA).

Article Title: EF1A interacting with nucleocapsid protein of transmissible gastroenteritis coronavirus and plays a role in virus replication
Article Snippet: .. PCR products were subcloned into a prokaryotic expression pGEX-6p-1 vector (GE Healthcare). .. Recombinant expression plasmid was designated as pGEX-TGEV-N and confirmed by DNA sequencing.

Article Title: Characterization of TgPuf1, a member of the Puf family RNA-binding proteins from Toxoplasma gondii
Article Snippet: .. Expression of recombinant TgPuf1 Puf domain in Escherichia coli To express the conserved RNA-binding domain of TgPuf1 in bacteria, PCR was performed with T. gondii cDNA using two primers (CGGGATCC AGAAAAGGCGACTCAAAAG and ATAAGAATGCGGCCGC GTCACTGAAACCTGAGATG) designed to clone at the Bam HI and Not I sites of the expression vector pGEX-6P-1 (GE Healthcare). ..

Article Title: Cloning and Characterization of a 2-Cys Peroxiredoxin from Babesiagibsoni
Article Snippet: .. The PCR product was digested with Eco RI andXho I and then ligated to the pGEX-6P1 vector containing an ORF encoding a glutathione S -transferase (GST)-fusion protein (GE Healthcare, Piscataway, NJ, U.S.A.). rBgTPx-1 was expressed as a GST-fusion protein in E. coli and purified using Glutathione-Sepharose 4B beads (GE Healthcare) as previously described [ ]. .. Then, GST was cleaved by PreScission protease (GE Healthcare) according to the manufacturer’s instructions.

Article Title: Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants
Article Snippet: .. GST-tagged TKTKK1 construction and Western blot hybridization The GST -tagged pGEX-6P-1 vector (GE Healthcare Life Sciences) was used for sub-cloning TKTKK1 by fusing with the GST sequence at its 3′-terminal. .. After verification by sequencing, the new plasmid pGEX-6P-1 with GST-TKTKK1 was transformed into the E. coli BL21.

Article Title: A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma
Article Snippet: .. Briefly, Rosetta (DE3) Escherichia coli cells were transformed with the pGEX-6P-1-GST vector or pGEX-6P-1-GST-PFKL, and then, expression was induced using 0.5 mM IPTG at 16 °C for 16 h. The E. coli were lysed, and the extracts were incubated with glutathione–Sepharose 4B beads (17075601; GE Healthcare Biosciences AB) at 4 °C for 1 h. The beads were then incubated with purified GFP-tagged A20, which were prepared through IP, for an additional 4 h. Proteins that had interacted were eluted in elution buffer (50 mM Tris-HCl pH 8.0 and 20 mM reduced glutathione) and were subjected to immunoblotting using anti-GFP antibody. .. Extracts from E. coli expressing only a GST tag were used as the negative control.

Article Title: A Second Lysine-Specific Serine Protease from Lysobacter sp. Strain IB-9374
Article Snippet: .. The insert DNA was digested with BamHI and EcoRI and then ligated into an expression vector of GST fusion protein, pGEX-6P-1 (Amersham Pharmacia Biotech AB), and digested with the same restriction enzyme, giving pGEX-lepB (Fig. ). ..

Hybridization:

Article Title: Improving protein content and quality by over-expressing artificially synthetic fusion proteins with high lysine and threonine constituent in rice plants
Article Snippet: .. GST-tagged TKTKK1 construction and Western blot hybridization The GST -tagged pGEX-6P-1 vector (GE Healthcare Life Sciences) was used for sub-cloning TKTKK1 by fusing with the GST sequence at its 3′-terminal. .. After verification by sequencing, the new plasmid pGEX-6P-1 with GST-TKTKK1 was transformed into the E. coli BL21.

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    GE Healthcare gst tagged gli1 deletion proteins
    <t>GLI1</t> interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e <t>GST</t> pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6
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    GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Journal: Communications Biology

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    doi: 10.1038/s42003-018-0275-4

    Figure Lengend Snippet: GLI1 interacts with the MEP50/PRMT5 complex. a FLAG-GLI1 interacted with endogenous MEP50 and interaction of FLAG-GLI1 and MEP50 was increased by HH signalling pathway activation. C3H10T1/2 cells were transfected with FLAG-GLI1 or the empty vector for 24 h and then treated with 300 nM SAG for an additional 24 h. Interaction of FLAG-GLI1 and MEP50 was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-MEP50 antibodies. b Schematic structures of MEP50 deletion mutants. c Mapping of the GLI1-binding region in MEP50 by immunoprecipitation analysis. HEK293T cells were transfected with Myc-MEP50 deletion mutants and FLAG-GLI1 plasmids for 24 h. Interaction of FLAG-GLI1 and Myc-MEP50 deletion mutants was detected by immunoprecipitation with anti-FLAG antibody followed by immunoblot analysis using anti-FLAG and anti-Myc antibodies. d Schematic of GLI1 deletion mutants. e GST pull-down assays to map the MEP50-binding region in GLI1. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated Myc-MEP50 from HEK293T cells. Immunoblotting was performed with an anti-Myc antibody. In a and e , data represent one of three independent experiments with similar results. In c , data represent one of two independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Article Snippet: To generate GST-tagged GLI1 deletion proteins, GLI1 cDNA fragments were subcloned by PCR and inserted into the pGEX-6P-1 vector (GE Healthcare).

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Binding Assay, Incubation

    MEP50/PRMT5 complex induces GLI1 methylation. a , b Methylation of GLI1 in MEP50- ( a ) or PRMT5- ( b ) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in ( c ). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. 4 . In c , data represent one of three independent experiments with similar results. In a and d , data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Journal: Communications Biology

    Article Title: MEP50/PRMT5-mediated methylation activates GLI1 in Hedgehog signalling through inhibition of ubiquitination by the ITCH/NUMB complex

    doi: 10.1038/s42003-018-0275-4

    Figure Lengend Snippet: MEP50/PRMT5 complex induces GLI1 methylation. a , b Methylation of GLI1 in MEP50- ( a ) or PRMT5- ( b ) knockdown C3H10T1/2 cells. siMEP50-m2 and siPRMT5-m2 siRNAs were stably expressed by recombinant retroviruses. Cells transfected with FLAG-GLI1 were cultured for 24 h, followed by treatment with 300 nM SAG for 24 h. Methylated GLI1 was detected by immunoprecipitation with an anti-FLAG antibody followed by immunoblot with anti-SYM11 antibody. c In vitro methylation assays to determine the region including methylated arginine residues in GLI1 deletion mutants. HA-PRMT5 expression plasmid was transfected into HEK293T cells. At 48 h after transfection, the cells were lysed, and HA-PRMT5 was immunoprecipitated using an anti-HA (3F10) antibody. GST-GLI1 deletion mutants coupled to glutathione sepharose were incubated with immunoprecipitated HA-PRMT5 from HEK293T cells. Upper panel represents the methylated GST-GLI1 deletion mutant. Lower panel represents 20% input of GST-GLI1 deletion mutants detected by CBB R-250 staining. HA-PRMT5 expressed in 10% of total lysate used for immunoprecipitation is shown in the right panel. d In vitro methylation assays to determine methylation sites in GLI1 using amino acid substitutions (arginine to lysine) of candidate methylation sites. In vitro methylation assays were performed as described in ( c ). Upper panel represents methylated GST-GLI1 mutants. Lower panel represents 20% input of GST-GLI1 mutants detected by CBB R-250 staining. Underlined text denotes highly conserved residues among mammals, as shown in Supplementary Fig. 4 . In c , data represent one of three independent experiments with similar results. In a and d , data represent one of twice independent experiments with similar results. Unprocessed original scans of blots are shown in Supplementary Fig. 6

    Article Snippet: To generate GST-tagged GLI1 deletion proteins, GLI1 cDNA fragments were subcloned by PCR and inserted into the pGEX-6P-1 vector (GE Healthcare).

    Techniques: Methylation, Stable Transfection, Recombinant, Transfection, Cell Culture, Immunoprecipitation, In Vitro, Expressing, Plasmid Preparation, Incubation, Mutagenesis, Staining