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Transcript levels of cat in B. burgdorferi B31-A3 as measured by <t>QRT-PCR.</t> All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.
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1) Product Images from "Insights into the complex regulation of rpoS in Borrelia burgdorferi"

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

Journal: Molecular Microbiology

doi: 10.1111/j.1365-2958.2007.05813.x

Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.
Figure Legend Snippet: Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

Techniques Used: Quantitative RT-PCR, Standard Deviation, Plasmid Preparation

Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

Techniques Used: Quantitative RT-PCR, Standard Deviation

Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.
Figure Legend Snippet: Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

Techniques Used: Quantitative RT-PCR, Standard Deviation

Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.
Figure Legend Snippet: Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

Techniques Used: Quantitative RT-PCR, Standard Deviation

Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.
Figure Legend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

Techniques Used: Quantitative RT-PCR, Standard Deviation

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

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

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.338426

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

Techniques Used: Expressing, Isolation, Quantitative RT-PCR

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

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

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl852

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

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

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

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

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

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

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

doi:

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

Techniques Used: Polymerase Chain Reaction, Amplification

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

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

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

Techniques Used: Labeling, Polymerase Chain Reaction

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

Techniques Used: Labeling, Polymerase Chain Reaction, Mutagenesis

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

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

Journal: Infection and Immunity

doi: 10.1128/IAI.70.6.3234-3248.2002

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

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

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

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

Journal: PLoS ONE

doi: 10.1371/journal.pone.0099028

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

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

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

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

Journal: Human pathology

doi: 10.1016/j.humpath.2012.01.019

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

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

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

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

Journal: Journal of Medical Microbiology

doi: 10.1099/jmm.0.040055-0

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

Techniques Used: Polymerase Chain Reaction, Amplification

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

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

Journal: Journal of Virology

doi: 10.1128/JVI.01149-15

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

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

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

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

Journal: Journal of Virology

doi: 10.1128/JVI.00694-11

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

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

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

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

Journal: Inflammatory bowel diseases

doi: 10.1002/ibd.21144

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

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

12) Product Images from "Mice Lacking Hbp1 Function Are Viable and Fertile"

Article Title: Mice Lacking Hbp1 Function Are Viable and Fertile

Journal: PLoS ONE

doi: 10.1371/journal.pone.0170576

Immunohistochemical and gene expression analysis of Hbp1 +/+ and Hbp1 -/- embryonic gonads. (A) Proliferation marker Ki67 in germ cells (MVH-positive cells) was detected in both Hbp1 +/+ and Hbp1 -/- gonads at 12.5 dpc, but was absent in germ cells of 14.5 and 16.5 dpc gonads of both genotypes. Pluripotency marker OCT3/4 was expressed similarly in germ cells (MVH-positive cells) in Hbp1 +/+ and Hbp1 -/- gonads at 14.5 dpc and was undetectable at 16.5 dpc. Scale bar = 50μm. (B) qRT-PCR analysis of Hbp1 +/+ and Hbp1 -/- 16.5 dpc gonad samples revealed comparable expression between of various germ cell and somatic cell markers. Germ cell markers: Mvh , Oct3/4 and G 1 /G 0 arrest indicator p63 , somatic cell markers Fgf9 and Sox9 , Retinoblastoma family members Rb1 , p130 and p107 , and cell cycle regulators p21 , Ccnd1-3 and p53 displayed no significant difference between Hbp1 +/- and Hbp1 -/- samples. Samples normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate). * P
Figure Legend Snippet: Immunohistochemical and gene expression analysis of Hbp1 +/+ and Hbp1 -/- embryonic gonads. (A) Proliferation marker Ki67 in germ cells (MVH-positive cells) was detected in both Hbp1 +/+ and Hbp1 -/- gonads at 12.5 dpc, but was absent in germ cells of 14.5 and 16.5 dpc gonads of both genotypes. Pluripotency marker OCT3/4 was expressed similarly in germ cells (MVH-positive cells) in Hbp1 +/+ and Hbp1 -/- gonads at 14.5 dpc and was undetectable at 16.5 dpc. Scale bar = 50μm. (B) qRT-PCR analysis of Hbp1 +/+ and Hbp1 -/- 16.5 dpc gonad samples revealed comparable expression between of various germ cell and somatic cell markers. Germ cell markers: Mvh , Oct3/4 and G 1 /G 0 arrest indicator p63 , somatic cell markers Fgf9 and Sox9 , Retinoblastoma family members Rb1 , p130 and p107 , and cell cycle regulators p21 , Ccnd1-3 and p53 displayed no significant difference between Hbp1 +/- and Hbp1 -/- samples. Samples normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate). * P

Techniques Used: Immunohistochemistry, Expressing, Marker, Quantitative RT-PCR

Hbp1 expression in Rb +/+ and Rb -/- mutant XY gonads. Using qRT-PCR analysis, normalizing gene expression to 18S RNA, both Fl-Hbp1 (A) and ΔHbp1 (B) gene expression was significantly increased in Rb -/- mutant XY gonads at 14.5 dpc. In 16.5 dpc Rb -/- cultured XY gonads, there was no significant difference in either Fl-Hbp1 or ΔHbp1 gene expression, although germ cell marker Mvh was significantly increased at this timepoint ( C ). When gene expression was normalized to germ cell marker Mvh , both Fl-Hbp1 (D) and ΔHbp1 (E) gene expression was significantly decreased in 16.5 dpc Rb -/- cultured XY gonads. (mean ± S.E.M of three independent experiments, each performed in triplicate; wildtype controls ( Rb +/+ ) set to 1). * P
Figure Legend Snippet: Hbp1 expression in Rb +/+ and Rb -/- mutant XY gonads. Using qRT-PCR analysis, normalizing gene expression to 18S RNA, both Fl-Hbp1 (A) and ΔHbp1 (B) gene expression was significantly increased in Rb -/- mutant XY gonads at 14.5 dpc. In 16.5 dpc Rb -/- cultured XY gonads, there was no significant difference in either Fl-Hbp1 or ΔHbp1 gene expression, although germ cell marker Mvh was significantly increased at this timepoint ( C ). When gene expression was normalized to germ cell marker Mvh , both Fl-Hbp1 (D) and ΔHbp1 (E) gene expression was significantly decreased in 16.5 dpc Rb -/- cultured XY gonads. (mean ± S.E.M of three independent experiments, each performed in triplicate; wildtype controls ( Rb +/+ ) set to 1). * P

Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Cell Culture, Marker

Detection of Fl-Hbp1 and ΔHbp1 transcripts in wildtype and W e /W e gonads. (A) Detection of Fl-Hbp1 and ΔHbp1 in 14.5 dpc XX and XY gonads using whole mount in situ hybridisation (B) qRT-PCR analysis detected lower levels of Fl-Hbp1 and ΔHbp1 gene expression in 13.5 dpc XY and XX W e /W e mutant gonads which lack germ cells. Expression was normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate) and wildtype controls set to 1. * P
Figure Legend Snippet: Detection of Fl-Hbp1 and ΔHbp1 transcripts in wildtype and W e /W e gonads. (A) Detection of Fl-Hbp1 and ΔHbp1 in 14.5 dpc XX and XY gonads using whole mount in situ hybridisation (B) qRT-PCR analysis detected lower levels of Fl-Hbp1 and ΔHbp1 gene expression in 13.5 dpc XY and XX W e /W e mutant gonads which lack germ cells. Expression was normalised to 18S RNA (mean ± S.E.M of three independent experiments, each performed in triplicate) and wildtype controls set to 1. * P

Techniques Used: In Situ, Hybridization, Quantitative RT-PCR, Expressing, Mutagenesis

Alternative splicing of Hbp1 transcripts. (A) Gene structure of the two Hbp1 splice variants. The full-length transcript comprises 11 exons and the truncated transcript is composed of the first 9 exons. Both transcripts contain identical 5’UTRs in addition to distinct 3’UTRs (open boxes), the specific riboprobes and real time PCR probes are depicted. (B) Location of HBP1 protein domains and nuclear localisation signals (NLS).
Figure Legend Snippet: Alternative splicing of Hbp1 transcripts. (A) Gene structure of the two Hbp1 splice variants. The full-length transcript comprises 11 exons and the truncated transcript is composed of the first 9 exons. Both transcripts contain identical 5’UTRs in addition to distinct 3’UTRs (open boxes), the specific riboprobes and real time PCR probes are depicted. (B) Location of HBP1 protein domains and nuclear localisation signals (NLS).

Techniques Used: Real-time Polymerase Chain Reaction

13) Product Images from "Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors"

Article Title: Malaria parasite DNA-harbouring vesicles activate cytosolic immune sensors

Journal: Nature Communications

doi: 10.1038/s41467-017-02083-1

P. falciparum EV-DNA activates STING-dependent signaling in monocytes. a . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage-derived or uRBC-derived vesicles for 1, 6 and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05. b . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage or uRBC-derived vesicles for 1, 6 and 24 h. An ELISA assay was performed for CCL5 and CXCL10. HEK blue IFNα/β was performed. SD and T -test analysis * p ≤ 0.05. c . THP-1 cells were incubated with P. falciparum ring-stage-derived vesicles, P. falciparum gDNA or transfected with poly(dA:dT) for 24 h. WB analysis was performed for STING, pIRF3, pTBK1 and α actin, sm-size marker. d . THP-1 cells were incubated with P. falciparum ring stage-derived vesicles for 24 h. Confocal microscopy images were taken for STING, pIRF3, pTBK1 (FITC), and DAPI. Scale bar 10 μm
Figure Legend Snippet: P. falciparum EV-DNA activates STING-dependent signaling in monocytes. a . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage-derived or uRBC-derived vesicles for 1, 6 and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05. b . THP-1 or STING KO THP-1 cells were incubated with P. falciparum ring-stage or uRBC-derived vesicles for 1, 6 and 24 h. An ELISA assay was performed for CCL5 and CXCL10. HEK blue IFNα/β was performed. SD and T -test analysis * p ≤ 0.05. c . THP-1 cells were incubated with P. falciparum ring-stage-derived vesicles, P. falciparum gDNA or transfected with poly(dA:dT) for 24 h. WB analysis was performed for STING, pIRF3, pTBK1 and α actin, sm-size marker. d . THP-1 cells were incubated with P. falciparum ring stage-derived vesicles for 24 h. Confocal microscopy images were taken for STING, pIRF3, pTBK1 (FITC), and DAPI. Scale bar 10 μm

Techniques Used: Incubation, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Marker, Confocal Microscopy

P. falciparum DNA stimulates innate immune gene induction in monocytes. a THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. RT–PCR was performed for the products IFNα , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. b THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. c THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05. d THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05
Figure Legend Snippet: P. falciparum DNA stimulates innate immune gene induction in monocytes. a THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. RT–PCR was performed for the products IFNα , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. b THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. RT–PCR was performed for IFNA , IFNB , CXCL10 , IFIT1 and CCL5 . SD and T -test analysis * p ≤ 0.05, ** p ≤ 0.01. c THP-1 cells were transfected or incubated with P. falciparum genomic DNA for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05. d THP-1 cells were transfected or incubated with poly(dA:dT) for 1, 6, and 24 h. ELISA were performed for CXCL10 and CCL5 and HEK blue IFNα/β. SD and T -test analysis * p ≤ 0.05

Techniques Used: Transfection, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

DNA-binding proteins and mitochondrial and apicoplast genes in EVs. a PCR for Ev-DNA markers, apicoplast ( ssu-api ), mitochondria ( ssud ) and nuclear genes ( msp2 , rap14 , and gap40 ). Plasmid control PuF-1 was added externally to EVs prior to DNase I treatment. b P. falciparum msp2 gene Ev-FISH images of ring and trophozoite EVs and uRBC vesicles. c P. falciparum ssu-api and ssud genes Ev-FISH of ring EVs. d H3 and H4 protein WB analysis for 2–5 OP gradient fractions (F). e P. falciparum H4 protein IFA. f P . falciparum parasites release EVs during the early post-invasion phase. Fluorescence microscopy using DAPI in EVs produced by iRBCs across their life cycle. Images of EVs collected at 12, 24, 36 and 48 h post invasion. Giemsa stains (first column) show the state of the parasites prior to collecting EVs at each time point. g SR1 control IFA
Figure Legend Snippet: DNA-binding proteins and mitochondrial and apicoplast genes in EVs. a PCR for Ev-DNA markers, apicoplast ( ssu-api ), mitochondria ( ssud ) and nuclear genes ( msp2 , rap14 , and gap40 ). Plasmid control PuF-1 was added externally to EVs prior to DNase I treatment. b P. falciparum msp2 gene Ev-FISH images of ring and trophozoite EVs and uRBC vesicles. c P. falciparum ssu-api and ssud genes Ev-FISH of ring EVs. d H3 and H4 protein WB analysis for 2–5 OP gradient fractions (F). e P. falciparum H4 protein IFA. f P . falciparum parasites release EVs during the early post-invasion phase. Fluorescence microscopy using DAPI in EVs produced by iRBCs across their life cycle. Images of EVs collected at 12, 24, 36 and 48 h post invasion. Giemsa stains (first column) show the state of the parasites prior to collecting EVs at each time point. g SR1 control IFA

Techniques Used: DNA Binding Assay, Polymerase Chain Reaction, Plasmid Preparation, Fluorescence In Situ Hybridization, Western Blot, Immunofluorescence, Fluorescence, Microscopy, Produced

14) Product Images from "Drosophila SPF45: A Bifunctional Protein with Roles in Both Splicing and DNA Repair"

Article Title: Drosophila SPF45: A Bifunctional Protein with Roles in Both Splicing and DNA Repair

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.0020178

Sxl Splicing Is Compromised in a spf45 Loss-of-Function Background (A) Synergistic genetic interactions between Sxl and spf45 J23 leads to female lethality. In these assays females of the indicated genotype were mated to either Sxl f1 /Y males or Sxl + /Y control males and the resulting progeny scored. On the assumption that an equal number of male and female progeny should be generated from each cross, the percent female viability was calculated by comparing the number of females recovered with the number of males recovered. (B) Diagram of the Sxl reporter construct that mirrors native Sxl splicing in all tissues tested. The arrows below the construct indicate the position of the PCR pairs used for RT–PCR. (C) Synergistic lethal interactions between Sxl and spf45 J23 are correlated with Sxl splicing defects. Splicing was assayed by an RT–PCR-based assay using RNA isolated from a pool of embryos in which only the female embryos carry the reporter construct (lanes 3 and 4). In lane 4, this pool of embryos was collected from spf45 J23 homozygous females crossed to males carrying an X chromosome that carries both Sxl f1 and a copy of the Sxl reporter construct. Controls include splicing of the reporter construct in adult males (lane 1), splicing of the reporter construct in adult females (lane 2), and Sxl + embryos collected from spf45 J23 homozygous mothers (lane 3).
Figure Legend Snippet: Sxl Splicing Is Compromised in a spf45 Loss-of-Function Background (A) Synergistic genetic interactions between Sxl and spf45 J23 leads to female lethality. In these assays females of the indicated genotype were mated to either Sxl f1 /Y males or Sxl + /Y control males and the resulting progeny scored. On the assumption that an equal number of male and female progeny should be generated from each cross, the percent female viability was calculated by comparing the number of females recovered with the number of males recovered. (B) Diagram of the Sxl reporter construct that mirrors native Sxl splicing in all tissues tested. The arrows below the construct indicate the position of the PCR pairs used for RT–PCR. (C) Synergistic lethal interactions between Sxl and spf45 J23 are correlated with Sxl splicing defects. Splicing was assayed by an RT–PCR-based assay using RNA isolated from a pool of embryos in which only the female embryos carry the reporter construct (lanes 3 and 4). In lane 4, this pool of embryos was collected from spf45 J23 homozygous females crossed to males carrying an X chromosome that carries both Sxl f1 and a copy of the Sxl reporter construct. Controls include splicing of the reporter construct in adult males (lane 1), splicing of the reporter construct in adult females (lane 2), and Sxl + embryos collected from spf45 J23 homozygous mothers (lane 3).

Techniques Used: Generated, Construct, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Isolation

15) Product Images from "The Serotonin 5-HT7Dro Receptor Is Expressed in the Brain of Drosophila, and Is Essential for Normal Courtship and Mating"

Article Title: The Serotonin 5-HT7Dro Receptor Is Expressed in the Brain of Drosophila, and Is Essential for Normal Courtship and Mating

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020800

dsRNAi construct effectively reduces 5-HT 7 Dro transcript levels. RNA from the heads of male flies carrying either the sym-p5-HT 7 RNAi (white box), the 5-HT 7 Dro-GAL4 (gray box), or both (F1, black box) transgenes was used in quantitative real-time PCR to examine 5-HT 7 Dro gene expression. Flies carrying both transcripts show an approximately 80% decrease in 5-HT 7 Dro transcript levels. Reactions were performed in quadruplicate for each gene. RpL32 expression was used as the reference control to normalize expression between treatment groups (Error bars indicate SEM).
Figure Legend Snippet: dsRNAi construct effectively reduces 5-HT 7 Dro transcript levels. RNA from the heads of male flies carrying either the sym-p5-HT 7 RNAi (white box), the 5-HT 7 Dro-GAL4 (gray box), or both (F1, black box) transgenes was used in quantitative real-time PCR to examine 5-HT 7 Dro gene expression. Flies carrying both transcripts show an approximately 80% decrease in 5-HT 7 Dro transcript levels. Reactions were performed in quadruplicate for each gene. RpL32 expression was used as the reference control to normalize expression between treatment groups (Error bars indicate SEM).

Techniques Used: Construct, Real-time Polymerase Chain Reaction, Expressing

16) Product Images from "Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells"

Article Title: Transcription factor Ebf1 regulates differentiation stage-specific signaling, proliferation, and survival of B cells

Journal: Genes & Development

doi: 10.1101/gad.187328.112

Impaired B lymphopoiesis and expression of regulatory genes in Ebf1 fl/fl RERT Cre bone marrow. ( A ) Immunoblot analysis to detect Ebf1 and Stat5a in lysates of splenic B cells from mice that were treated with tamoxifen 4-OHT (3 mg) on two consecutive days and sacrificed on various days after first administration. ( B ) Flow cytometric analysis of bone marrow to detect B220 + CD43 + pro-B cells, B220 intermediate CD43 − pre-B cells, and B220 high CD43 − recirculating B cells. Numbers in quadrants indicate percentage of cells. ( C ) Flow cytometric analysis to detect differentiation and generation of CD25 + κ − pre-B cells and CD25 − κ + immature B cells from CD19 + CD43 + c-kit − pro-B cells in fetal liver cell cultures. Cells cultured in the presence of OP9 feeders and IL-7 were treated with 2 μM 4-OHT for 24 h and cultured for an additional 4 d. Differentiation was induced by withdrawal of IL-7. ( D ) Semiquantitative RT–PCR analysis of κ light chain GLTs in pro-B cells that were cultured in the presence of OP9 feeders and IL-7 for 6 d and treated with 2 μM 4-OHT during the first day of culture. Data are representative of at least four ( A ) or three ( B – D ) experiments. ( E ) Quantitative RT–PCR analysis to examine the expression of B-cell-specific regulatory genes in pro-B-cell cultures. Data represent mean values of three independent biological replicates, and the raw cycle values were normalized to actin expression. Error bars indicate standard deviation (SD). ( F ) Quantification of Ebf1 binding to target genes in 38B9 cells by ChIP and quantitative PCR analysis. Binding is represented as percentage of input chromatin, and error bars represent SD of duplicate ChIP experiments (see also Supplemental Fig. S1; Supplemental Table S1). ( G ) Sequence tag profiles in pro-B cells and splenic B cells at the Irf4 and Irf8 ) and splenic B cells (this study), as well as DNase I hypersensitivity sites (DHS) in CD19 + ) and splenic B cells (this study).
Figure Legend Snippet: Impaired B lymphopoiesis and expression of regulatory genes in Ebf1 fl/fl RERT Cre bone marrow. ( A ) Immunoblot analysis to detect Ebf1 and Stat5a in lysates of splenic B cells from mice that were treated with tamoxifen 4-OHT (3 mg) on two consecutive days and sacrificed on various days after first administration. ( B ) Flow cytometric analysis of bone marrow to detect B220 + CD43 + pro-B cells, B220 intermediate CD43 − pre-B cells, and B220 high CD43 − recirculating B cells. Numbers in quadrants indicate percentage of cells. ( C ) Flow cytometric analysis to detect differentiation and generation of CD25 + κ − pre-B cells and CD25 − κ + immature B cells from CD19 + CD43 + c-kit − pro-B cells in fetal liver cell cultures. Cells cultured in the presence of OP9 feeders and IL-7 were treated with 2 μM 4-OHT for 24 h and cultured for an additional 4 d. Differentiation was induced by withdrawal of IL-7. ( D ) Semiquantitative RT–PCR analysis of κ light chain GLTs in pro-B cells that were cultured in the presence of OP9 feeders and IL-7 for 6 d and treated with 2 μM 4-OHT during the first day of culture. Data are representative of at least four ( A ) or three ( B – D ) experiments. ( E ) Quantitative RT–PCR analysis to examine the expression of B-cell-specific regulatory genes in pro-B-cell cultures. Data represent mean values of three independent biological replicates, and the raw cycle values were normalized to actin expression. Error bars indicate standard deviation (SD). ( F ) Quantification of Ebf1 binding to target genes in 38B9 cells by ChIP and quantitative PCR analysis. Binding is represented as percentage of input chromatin, and error bars represent SD of duplicate ChIP experiments (see also Supplemental Fig. S1; Supplemental Table S1). ( G ) Sequence tag profiles in pro-B cells and splenic B cells at the Irf4 and Irf8 ) and splenic B cells (this study), as well as DNase I hypersensitivity sites (DHS) in CD19 + ) and splenic B cells (this study).

Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing

Roles of Ebf1 in peripheral B-cell subsets and BAFF-R-mediated cell survival. ( A ) Flow cytometric analysis of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre splenocytes to detect IgM hi IgD + immature B cells and IgM hi IgD hi mature B cells ( top panels), IgM hi IgD hi B220 + AA4.1 + transitional B cells ( middle panels), and B220 + CD23 − Cd21 hi MZ B cells and B220 + CD23 + CD21 + FO B cells ( bottom panels). Numbers in quadrants indicate percentage of cells. ( B ) Flow cytometric analysis of Ebf1 +/fl Cd21 Cre and Ebf1 fl/fl Cd21 Cre splenocytes to detect MZ B cells and FO B cells. ( C ) Quantitative RT–PCR analysis of Ebf1-regulated targets in sorted MZ B cells. Fold expression values are relative to heterozygote control cells. ( D ) ChIP analysis to examine binding of Ebf1 to the Tnfrsf13c (Baff-R) locus in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. ( E ) Flow cytometric analysis of proliferation of splenic B cells that were depleted of non-FO B cells with antibodies directed against CD43, CD4, CD8a, Gr1, AA4.1, and CD9. Cells sorted 10 d after the initial 4-OHT treatment were CFSE-labeled and stimulated with LPS or αIgM F(ab′) 2 . Proliferation was determined by CFSE dilution 3 d after stimulation. Numbers indicate percentages of cells in the area marked by the thin bar. ( F ) Analysis of apoptosis of LPS- or α-IgM-stimulated splenic B cells at the specified days (d) ( n = 3). ( G ) Flow cytometric analysis of surface expression of BAFF-R in resting and stimulated splenic B cells. Cells were activated with the indicated stimuli for 40 h. The graph is representative of three experiments. ( H ) Analysis of the survival of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre resting splenic B cells in the presence of optimal (20 ng/mL) and limiting (2 ng/mL) exogenous BAFF ( n = 3).
Figure Legend Snippet: Roles of Ebf1 in peripheral B-cell subsets and BAFF-R-mediated cell survival. ( A ) Flow cytometric analysis of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre splenocytes to detect IgM hi IgD + immature B cells and IgM hi IgD hi mature B cells ( top panels), IgM hi IgD hi B220 + AA4.1 + transitional B cells ( middle panels), and B220 + CD23 − Cd21 hi MZ B cells and B220 + CD23 + CD21 + FO B cells ( bottom panels). Numbers in quadrants indicate percentage of cells. ( B ) Flow cytometric analysis of Ebf1 +/fl Cd21 Cre and Ebf1 fl/fl Cd21 Cre splenocytes to detect MZ B cells and FO B cells. ( C ) Quantitative RT–PCR analysis of Ebf1-regulated targets in sorted MZ B cells. Fold expression values are relative to heterozygote control cells. ( D ) ChIP analysis to examine binding of Ebf1 to the Tnfrsf13c (Baff-R) locus in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. ( E ) Flow cytometric analysis of proliferation of splenic B cells that were depleted of non-FO B cells with antibodies directed against CD43, CD4, CD8a, Gr1, AA4.1, and CD9. Cells sorted 10 d after the initial 4-OHT treatment were CFSE-labeled and stimulated with LPS or αIgM F(ab′) 2 . Proliferation was determined by CFSE dilution 3 d after stimulation. Numbers indicate percentages of cells in the area marked by the thin bar. ( F ) Analysis of apoptosis of LPS- or α-IgM-stimulated splenic B cells at the specified days (d) ( n = 3). ( G ) Flow cytometric analysis of surface expression of BAFF-R in resting and stimulated splenic B cells. Cells were activated with the indicated stimuli for 40 h. The graph is representative of three experiments. ( H ) Analysis of the survival of Ebf1 +/fl RERT Cre and Ebf1 fl/fl RERT Cre resting splenic B cells in the presence of optimal (20 ng/mL) and limiting (2 ng/mL) exogenous BAFF ( n = 3).

Techniques Used: Flow Cytometry, Quantitative RT-PCR, Expressing, Chromatin Immunoprecipitation, Binding Assay, Magnetic Cell Separation, Labeling

Ebf1 regulates proliferative expansion and survival of pro-B cells. ( A ) Analysis of apoptosis of pro-B cells cultured in the presence of feeders and IL-7 after induced deletion of Ebf1 . Numbers of viable (annexin V- and PI-negative) cells were determined by flow cytometry at the indicated time points after 4-OHT treatment, and the data were converted to the percentage of viable cells at day 3. Line graphs represent the average survival of three independent cell cultures at various days (d). ( B , C ) Analysis of survival of primary Ebf1 fl/fl RERT Cre TgBcl2 pro-B cells ( n = 3) and transformed Ebf1 fl/fl RERT Cre ::A-MuLV pro-B cells ( n = 4), as described above. ( D , E ) Analysis of apoptosis in A-MuLV-transformed Ebf1 fl/fl pro-B cells that have been transduced with a GFP-expressing retrovirus or bicistronic retroviruses expressing both GFP and various Ebf1 target genes. Cells sorted for GFP expression were treated with 4-OHT, and the percentages of viable cells were determined at the indicated time points after treatment ( n = 4). Full and partial rescue of the survival defect of Ebf1-deficient cells is observed by expression of c-Myb and Bcl2l1, respectively. ( F ) Flow cytometric analysis of the proliferation of Ebf1 +/fl RERT Cre (thick line) and Ebf1 fl/fl RERT Cre (gray fill) pro-B cells treated with 2 μM 4-OHT for 24 h, cultured for 2 d without 4-OHT, labeled with CFSE, and assessed by CFSE dilution after 3 d ( n = 4). Numbers indicate the percentage of cells in the area marked by the thin bar. ( G , H ) Flow cytometric analysis of cell cycle progression of Ebf1 +/fl RERT Cre (black fill) and Ebf1 fl/fl RERT Cre (gray fill) primary pro-B ( G ) or A-MuLV-transformed pro-B ( H ) cell cultures 5 d after 4-OHT administration. Cells were fixed, stained with PI to assess DNA content, and gated for intact cells. Representative plot and quantitative analysis of cell cycle distribution are indicated ( n = 3). Numbers indicate percentage of cells. ( I ) Quantitative RT–PCR analysis to determine the expression of regulatory genes involved in cell cycle or survival and identified as indirect or direct (*) Ebf1 targets. Primary pro-B cells ( top panel) and A-MuLV-transformed pro-B cells ( bottom panel) were harvested 5 d after 4-OHT treatment. Data represent mean values of three biological replicates, and raw cycle values were normalized to actin . Ebf1 +/+ samples were set to 100%. ( J , K ) Immunoblot analysis to detect Cdc2 phosphorylation on Tyr 15 in primary ( J ) and A-MuLV-transformed ( K ) Ebf1 fl/fl RERT Cre pro-B cells. ( L ) Immunoblot analysis to detect Rb phosphorylation and expression of Ebf1, c-Myb, Cdc6, and Cdt1 in A-MuLV-transformed Ebf1 fl/fl RERT Cre pro-B cells.
Figure Legend Snippet: Ebf1 regulates proliferative expansion and survival of pro-B cells. ( A ) Analysis of apoptosis of pro-B cells cultured in the presence of feeders and IL-7 after induced deletion of Ebf1 . Numbers of viable (annexin V- and PI-negative) cells were determined by flow cytometry at the indicated time points after 4-OHT treatment, and the data were converted to the percentage of viable cells at day 3. Line graphs represent the average survival of three independent cell cultures at various days (d). ( B , C ) Analysis of survival of primary Ebf1 fl/fl RERT Cre TgBcl2 pro-B cells ( n = 3) and transformed Ebf1 fl/fl RERT Cre ::A-MuLV pro-B cells ( n = 4), as described above. ( D , E ) Analysis of apoptosis in A-MuLV-transformed Ebf1 fl/fl pro-B cells that have been transduced with a GFP-expressing retrovirus or bicistronic retroviruses expressing both GFP and various Ebf1 target genes. Cells sorted for GFP expression were treated with 4-OHT, and the percentages of viable cells were determined at the indicated time points after treatment ( n = 4). Full and partial rescue of the survival defect of Ebf1-deficient cells is observed by expression of c-Myb and Bcl2l1, respectively. ( F ) Flow cytometric analysis of the proliferation of Ebf1 +/fl RERT Cre (thick line) and Ebf1 fl/fl RERT Cre (gray fill) pro-B cells treated with 2 μM 4-OHT for 24 h, cultured for 2 d without 4-OHT, labeled with CFSE, and assessed by CFSE dilution after 3 d ( n = 4). Numbers indicate the percentage of cells in the area marked by the thin bar. ( G , H ) Flow cytometric analysis of cell cycle progression of Ebf1 +/fl RERT Cre (black fill) and Ebf1 fl/fl RERT Cre (gray fill) primary pro-B ( G ) or A-MuLV-transformed pro-B ( H ) cell cultures 5 d after 4-OHT administration. Cells were fixed, stained with PI to assess DNA content, and gated for intact cells. Representative plot and quantitative analysis of cell cycle distribution are indicated ( n = 3). Numbers indicate percentage of cells. ( I ) Quantitative RT–PCR analysis to determine the expression of regulatory genes involved in cell cycle or survival and identified as indirect or direct (*) Ebf1 targets. Primary pro-B cells ( top panel) and A-MuLV-transformed pro-B cells ( bottom panel) were harvested 5 d after 4-OHT treatment. Data represent mean values of three biological replicates, and raw cycle values were normalized to actin . Ebf1 +/+ samples were set to 100%. ( J , K ) Immunoblot analysis to detect Cdc2 phosphorylation on Tyr 15 in primary ( J ) and A-MuLV-transformed ( K ) Ebf1 fl/fl RERT Cre pro-B cells. ( L ) Immunoblot analysis to detect Rb phosphorylation and expression of Ebf1, c-Myb, Cdc6, and Cdt1 in A-MuLV-transformed Ebf1 fl/fl RERT Cre pro-B cells.

Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Transformation Assay, Transduction, Expressing, Labeling, Staining, Quantitative RT-PCR

Ebf1 regulates γ3 and γ1 germline transcription by binding to the IgH 3′ Eα LCR. ( A ) Schematic representation of the IgH locus. Light-gray bars depict rearranged variable gene segments, dark gray bars represent enhancers, and gray circles represent the promoters (P) of the constant region (C) gene segments. The bottom part depicts a magnified view of the γ3 gene segment and indicates regulatory sequences, switch (S) region, and 3′ Eα LCR. DHSs are marked with arrows and numbers. Numbers in squares represent HSs that are directly bound by Ebf1. ( B ) Semiquantitative RT–PCR analysis of IgH constant region GLTs in unstimulated B cells and B cells stimulated with LPS + IL4 or LPS + TGFβ for 16 h. ( C ) ChIP analysis to examine binding of Ebf1 in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. (See also Supplemental Fig. S7.)
Figure Legend Snippet: Ebf1 regulates γ3 and γ1 germline transcription by binding to the IgH 3′ Eα LCR. ( A ) Schematic representation of the IgH locus. Light-gray bars depict rearranged variable gene segments, dark gray bars represent enhancers, and gray circles represent the promoters (P) of the constant region (C) gene segments. The bottom part depicts a magnified view of the γ3 gene segment and indicates regulatory sequences, switch (S) region, and 3′ Eα LCR. DHSs are marked with arrows and numbers. Numbers in squares represent HSs that are directly bound by Ebf1. ( B ) Semiquantitative RT–PCR analysis of IgH constant region GLTs in unstimulated B cells and B cells stimulated with LPS + IL4 or LPS + TGFβ for 16 h. ( C ) ChIP analysis to examine binding of Ebf1 in 38B9 pro-B cells or MACS-enriched splenic B cells. Binding is represented as percentage of input chromatin. (See also Supplemental Fig. S7.)

Techniques Used: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Magnetic Cell Separation

Impaired GC development in the spleens of Ebf1 fl/fl RERT Cre mice. ( A ) Flow cytometric analysis to detect GC B cells in the spleens of SRBC-immunized Ebf1 fl/fl RERT Cre and Ebf1 +/fl RERT Cre mice. Mice were immunized 8 d after the first 4-OHT treatment and were analyzed 6 d after immunization. Samples were gated on living B220 + cells, and the numbers on the FACS plots represent the percentage of PNA + FAS hi GC B cells ( n = 6). ( B ) Analysis of immune responses of Ebf1 fl/fl RERT Cre mice. Spleen sections were stained with PNA ( top panel) to reveal GC cells, and with antibodies to IgM (green) to reveal B-cell follicles and total IgG (green, bottom panel). Nuclei were stained with DAPI. ( C ) Gene expression analysis of sorted PNA + FAS hi GC B cells of the indicated genotypes by quantitative RT–PCR. Black and gray bars represent the averages of individually sorted samples ( n = 3). ( D ) Normal somatic hypermutation of Ebf1 fl/fl RERT Cre GC B cells upon immunization with SRBCs. Mice were immunized as described in A ; genomic DNA was extracted from sorted GC B cells and subjected to nested PCR amplification with two degenerate V and J segment primer pairs, and the amplified products corresponding to J H 3 were separated and cloned. The number of clones sequenced is shown in the circles in the middle of the pie diagrams; each diagram is representative of two independent experiments, with at least 40 cloned sequences each. ( E ) Relative titers of immunoglobulin subtypes in sera of Ebf1 +/fl RERT Cre mice (black triangles and blue lines represent individual samples and averages, respectively) and Ebf1 fl/fl RERT Cre mice (open circles and red lines represent individual samples and averages, respectively) at the specified days (d), determined by ELISA.
Figure Legend Snippet: Impaired GC development in the spleens of Ebf1 fl/fl RERT Cre mice. ( A ) Flow cytometric analysis to detect GC B cells in the spleens of SRBC-immunized Ebf1 fl/fl RERT Cre and Ebf1 +/fl RERT Cre mice. Mice were immunized 8 d after the first 4-OHT treatment and were analyzed 6 d after immunization. Samples were gated on living B220 + cells, and the numbers on the FACS plots represent the percentage of PNA + FAS hi GC B cells ( n = 6). ( B ) Analysis of immune responses of Ebf1 fl/fl RERT Cre mice. Spleen sections were stained with PNA ( top panel) to reveal GC cells, and with antibodies to IgM (green) to reveal B-cell follicles and total IgG (green, bottom panel). Nuclei were stained with DAPI. ( C ) Gene expression analysis of sorted PNA + FAS hi GC B cells of the indicated genotypes by quantitative RT–PCR. Black and gray bars represent the averages of individually sorted samples ( n = 3). ( D ) Normal somatic hypermutation of Ebf1 fl/fl RERT Cre GC B cells upon immunization with SRBCs. Mice were immunized as described in A ; genomic DNA was extracted from sorted GC B cells and subjected to nested PCR amplification with two degenerate V and J segment primer pairs, and the amplified products corresponding to J H 3 were separated and cloned. The number of clones sequenced is shown in the circles in the middle of the pie diagrams; each diagram is representative of two independent experiments, with at least 40 cloned sequences each. ( E ) Relative titers of immunoglobulin subtypes in sera of Ebf1 +/fl RERT Cre mice (black triangles and blue lines represent individual samples and averages, respectively) and Ebf1 fl/fl RERT Cre mice (open circles and red lines represent individual samples and averages, respectively) at the specified days (d), determined by ELISA.

Techniques Used: Mouse Assay, Flow Cytometry, FACS, Staining, Expressing, Quantitative RT-PCR, Nested PCR, Amplification, Clone Assay, Enzyme-linked Immunosorbent Assay

17) Product Images from "The NF-?B Factor RelB and Histone H3 Lysine Methyltransferase G9a Directly Interact to Generate Epigenetic Silencing in Endotoxin Tolerance *"

Article Title: The NF-?B Factor RelB and Histone H3 Lysine Methyltransferase G9a Directly Interact to Generate Epigenetic Silencing in Endotoxin Tolerance *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.000950

RelB, G9a, and HP1 form a complex at the silenced IL-1β promoter. A , a diagrammatic representation of the IL-1β proximal promoter region analyzed by ChIP. The NF-κB site and the location of the primers used in PCR are shown. B
Figure Legend Snippet: RelB, G9a, and HP1 form a complex at the silenced IL-1β promoter. A , a diagrammatic representation of the IL-1β proximal promoter region analyzed by ChIP. The NF-κB site and the location of the primers used in PCR are shown. B

Techniques Used: Chromatin Immunoprecipitation, Polymerase Chain Reaction

18) Product Images from "Generation and characterization of influenza A viruses with altered polymerase fidelity"

Article Title: Generation and characterization of influenza A viruses with altered polymerase fidelity

Journal: Nature communications

doi: 10.1038/ncomms5794

Replication kinetics of wild-type and PB1-V43I viruses under competition or suboptimal temperatures (a) One-step growth kinetics of wild-type and PB1-V43I H3N2 and H5N1 viruses using MOI 1–2 TCID 50 per cell in MDCK cells. Viral supernatants were collected every 2h post-infection and viral titres (mean ± SD log 10 TCID 50 per mL) from triplicates were shown. The replication kinetics has been repeated three times for the H3N2 wild-type and PB1-V43I viruses and once for the H5N1 wild-type and PB1-V43I viruses. (b) Competitive replication of wild-type and PB1-V43I mutant viruses in vitro . Wild-type and PB1-V43I viruses were premixed at different ratios prior to infection of MDCK cells. To determine the actual ratio in both the premixed (inoculum) and the viral supernatant after incubation for 2 days (progeny virus), clonal sequencing was performed to determine the ratio between Wuhan95 wild-type and PB1-V43I viruses. (c) As an alternative, in a separate experiment, instead of clonal sequencing, plaque assay was performed for the inoculums and passage-one viral supernatant after incubation for 2 days. Then, 32 clones were picked for each of the three inoculums and three corresponding passage-one viral cultures for viral RNA isolation and RT-PCR of the PB1 gene region that can distinguish wild-type virus from its V43I mutant counterpart. The actual ratio in the inoculums and the viral supernatant was shown in the same fashion. (d) Mean plaque sizes formed by the Wuhan95 wild-type and PB1-V43I viruses incubated at different temperatures. Wild-type or PB1-V43I mutant viruses were used to infect MDCK monolayers in triplicates and incubated under 0.5% agar overlay for 48 hours at 33°C, 37°C, or 39°C. The experiments were repeated independently twice and one representative result was shown. (e) Picture taken for the one representative plaque assay experiment performed for wild-type and V43I mutant viruses at the three temperatures. (f) Growth curve of wild-type and V43I mutant Wuhan95 viruses performed at 37°C, (g) 33°C, and (h) 39°C. For (f) to (h), viral titres (mean±SD log 10 TCID 50 per mL) from quadruplicated wells were shown. Three independent experiments were performed, with one representative experiment being displayed. P-values were based on Two-way ANOVA test with bonferroni post-tests. N.S., not statistically significant.
Figure Legend Snippet: Replication kinetics of wild-type and PB1-V43I viruses under competition or suboptimal temperatures (a) One-step growth kinetics of wild-type and PB1-V43I H3N2 and H5N1 viruses using MOI 1–2 TCID 50 per cell in MDCK cells. Viral supernatants were collected every 2h post-infection and viral titres (mean ± SD log 10 TCID 50 per mL) from triplicates were shown. The replication kinetics has been repeated three times for the H3N2 wild-type and PB1-V43I viruses and once for the H5N1 wild-type and PB1-V43I viruses. (b) Competitive replication of wild-type and PB1-V43I mutant viruses in vitro . Wild-type and PB1-V43I viruses were premixed at different ratios prior to infection of MDCK cells. To determine the actual ratio in both the premixed (inoculum) and the viral supernatant after incubation for 2 days (progeny virus), clonal sequencing was performed to determine the ratio between Wuhan95 wild-type and PB1-V43I viruses. (c) As an alternative, in a separate experiment, instead of clonal sequencing, plaque assay was performed for the inoculums and passage-one viral supernatant after incubation for 2 days. Then, 32 clones were picked for each of the three inoculums and three corresponding passage-one viral cultures for viral RNA isolation and RT-PCR of the PB1 gene region that can distinguish wild-type virus from its V43I mutant counterpart. The actual ratio in the inoculums and the viral supernatant was shown in the same fashion. (d) Mean plaque sizes formed by the Wuhan95 wild-type and PB1-V43I viruses incubated at different temperatures. Wild-type or PB1-V43I mutant viruses were used to infect MDCK monolayers in triplicates and incubated under 0.5% agar overlay for 48 hours at 33°C, 37°C, or 39°C. The experiments were repeated independently twice and one representative result was shown. (e) Picture taken for the one representative plaque assay experiment performed for wild-type and V43I mutant viruses at the three temperatures. (f) Growth curve of wild-type and V43I mutant Wuhan95 viruses performed at 37°C, (g) 33°C, and (h) 39°C. For (f) to (h), viral titres (mean±SD log 10 TCID 50 per mL) from quadruplicated wells were shown. Three independent experiments were performed, with one representative experiment being displayed. P-values were based on Two-way ANOVA test with bonferroni post-tests. N.S., not statistically significant.

Techniques Used: Infection, Mutagenesis, In Vitro, Incubation, Sequencing, Plaque Assay, Clone Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

19) Product Images from "DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation"

Article Title: DNA methylation-mediated silencing of PU.1 in leukemia cells resistant to cell differentiation

Journal: SpringerPlus

doi: 10.1186/2193-1801-2-392

Methylation status of the PU.1 promoter region. A) DNA chromatogram of the PU.1 upstream region using bisulfite-treated genomic DNA derived from MEL and MEL-R cell lines. Red circles highlight the cytosines of the four CpG islands of the MEL-R cell line that changed to thymines in the MEL-DS19 cell line after bisulfite treatment. B) Methylation analysis of the PCR products of the bisulfite-treated genomic DNA from A). The CG sequences were numbered according to (Shearstone et al. 2011 ) and their methylation status was determined.
Figure Legend Snippet: Methylation status of the PU.1 promoter region. A) DNA chromatogram of the PU.1 upstream region using bisulfite-treated genomic DNA derived from MEL and MEL-R cell lines. Red circles highlight the cytosines of the four CpG islands of the MEL-R cell line that changed to thymines in the MEL-DS19 cell line after bisulfite treatment. B) Methylation analysis of the PCR products of the bisulfite-treated genomic DNA from A). The CG sequences were numbered according to (Shearstone et al. 2011 ) and their methylation status was determined.

Techniques Used: Methylation, Derivative Assay, Polymerase Chain Reaction

DNA methylation status of the PU.1 locus regulatory region. A) A genomic map (not drawn to scale) depicts the regions containing the analyzed CpGs. The CpG colored in blue represent PCR amplified islands which however were not detected by the sequencing primers. CpG colored in red represent sequenced islands. B) Individual CpG methylation percentages of MEL DS19 and MEL-R cell lines.
Figure Legend Snippet: DNA methylation status of the PU.1 locus regulatory region. A) A genomic map (not drawn to scale) depicts the regions containing the analyzed CpGs. The CpG colored in blue represent PCR amplified islands which however were not detected by the sequencing primers. CpG colored in red represent sequenced islands. B) Individual CpG methylation percentages of MEL DS19 and MEL-R cell lines.

Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Amplification, Sequencing, CpG Methylation Assay

PCR probe to confirm the SFFV integration site within the PU.1 locus. A) Illustration of the multiple verification PCR amplifications using MEL genomic DNA as a template. Black arrows over the SFFV genome (gray box) indicate the pair of primers designed to amplify the SFFV-PU.1 junctions. Odd numbers 1 and 3 represent the primers used to identify the upstream integration junction and even numbers 2 and 4 represent the primers used for the downstream integration. Number 5 represents the long-range PCR (LR-PCR) used as a probe to confirm the complete SFFV integration. Black boxes correspond to the five exons of PU.1; the arrow above exon number one represents the initiation and direction of translation. B) Agarose gel electrophoresis performed using the primers schematized in A ) . C) Agarose gel electrophoresis of the LR-PCR probe to confirm SFFV integration; both the wild type (564 bp) and the integrated allele (6,859 bp) are visualized.
Figure Legend Snippet: PCR probe to confirm the SFFV integration site within the PU.1 locus. A) Illustration of the multiple verification PCR amplifications using MEL genomic DNA as a template. Black arrows over the SFFV genome (gray box) indicate the pair of primers designed to amplify the SFFV-PU.1 junctions. Odd numbers 1 and 3 represent the primers used to identify the upstream integration junction and even numbers 2 and 4 represent the primers used for the downstream integration. Number 5 represents the long-range PCR (LR-PCR) used as a probe to confirm the complete SFFV integration. Black boxes correspond to the five exons of PU.1; the arrow above exon number one represents the initiation and direction of translation. B) Agarose gel electrophoresis performed using the primers schematized in A ) . C) Agarose gel electrophoresis of the LR-PCR probe to confirm SFFV integration; both the wild type (564 bp) and the integrated allele (6,859 bp) are visualized.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

20) Product Images from "Mannose receptor regulates myoblast motility and muscle growth"

Article Title: Mannose receptor regulates myoblast motility and muscle growth

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200601102

MR is expressed in muscle cells during myoblast fusion. (A) Primary myoblasts (Mb) were induced to differentiate for 24 or 48 h. MR mRNA was analyzed by RT-PCR. Myogenin mRNA was assessed as a marker of myogenic differentiation. Phase-contrast images of muscle cells are shown to illustrate fusion progress at each time point. MR, 390 bp; Myogenin, 266 bp; 18S, 488 bp. (B) Representative images of muscle cells after 24 h of differentiation immunostained with an antibody against MR. Bar, 50 μm. (C) Primary myoblasts were differentiated for 24 h and subsequently treated with vehicle or 10 ng/ml IL-4 for 24 h. MR mRNA was analyzed by RT-PCR. (D) MR mRNA expression in WT or IL-4 receptor α-null (IL-4Rα −/− ) myotubes after 48 h in DM was examined by RT-PCR. MR, 390 bp; 18S, 488 bp. Representative ethidium bromide staining of agarose gels is shown with 18S ribosomal RNA as an internal control for all RT-PCR analyses. All data are indicative of results from three independent cell isolates.
Figure Legend Snippet: MR is expressed in muscle cells during myoblast fusion. (A) Primary myoblasts (Mb) were induced to differentiate for 24 or 48 h. MR mRNA was analyzed by RT-PCR. Myogenin mRNA was assessed as a marker of myogenic differentiation. Phase-contrast images of muscle cells are shown to illustrate fusion progress at each time point. MR, 390 bp; Myogenin, 266 bp; 18S, 488 bp. (B) Representative images of muscle cells after 24 h of differentiation immunostained with an antibody against MR. Bar, 50 μm. (C) Primary myoblasts were differentiated for 24 h and subsequently treated with vehicle or 10 ng/ml IL-4 for 24 h. MR mRNA was analyzed by RT-PCR. (D) MR mRNA expression in WT or IL-4 receptor α-null (IL-4Rα −/− ) myotubes after 48 h in DM was examined by RT-PCR. MR, 390 bp; 18S, 488 bp. Representative ethidium bromide staining of agarose gels is shown with 18S ribosomal RNA as an internal control for all RT-PCR analyses. All data are indicative of results from three independent cell isolates.

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

21) Product Images from "Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues"

Article Title: Ecdysone receptor directly binds the promoter of the Drosophila caspase dronc, regulating its expression in specific tissues

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200311057

Ecdysone-mediated dronc transcription is partially cycloheximide sensitive. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone (Ecd) for the indicated time in the presence or absence (Control) of 10 μg/ml cycloheximide (CHX). RNA extracted from cells was analyzed by RT-PCR. (B) Northern blot analysis was performed on RNA samples from cells treated as in A. Where indicated, cells were treated with cycloheximide (CHX) for 2 h.
Figure Legend Snippet: Ecdysone-mediated dronc transcription is partially cycloheximide sensitive. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone (Ecd) for the indicated time in the presence or absence (Control) of 10 μg/ml cycloheximide (CHX). RNA extracted from cells was analyzed by RT-PCR. (B) Northern blot analysis was performed on RNA samples from cells treated as in A. Where indicated, cells were treated with cycloheximide (CHX) for 2 h.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot

l(2)mbn cells express the EcR-B1 isoform, which binds to the dronc promoter. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone for the indicated time. RNA was extracted from cells and analyzed by RT-PCR to detect specific EcR isoforms. The last lane of the gel shows EcR isoforms expressed in late third instar larvae/prepupae (120 h after egg laying). (B) 9 μg of nuclear extracts prepared from l(2)mbn cells treated with ecdysone for 6 h was incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min in the presence of 2 μl of EcR common, EcR-B1, or EcR-A antibody. A mouse control antibody was also used. Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–Usp complex (EcR) and supershifted EcR–Usp complex (ss) are indicated.
Figure Legend Snippet: l(2)mbn cells express the EcR-B1 isoform, which binds to the dronc promoter. (A) 10 7 l(2)mbn cells were treated with 10 μM ecdysone for the indicated time. RNA was extracted from cells and analyzed by RT-PCR to detect specific EcR isoforms. The last lane of the gel shows EcR isoforms expressed in late third instar larvae/prepupae (120 h after egg laying). (B) 9 μg of nuclear extracts prepared from l(2)mbn cells treated with ecdysone for 6 h was incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min in the presence of 2 μl of EcR common, EcR-B1, or EcR-A antibody. A mouse control antibody was also used. Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–Usp complex (EcR) and supershifted EcR–Usp complex (ss) are indicated.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Incubation, Mutagenesis

dronc upstream promoter harbors a putative EcRBE. (A) In vitro translated EcR and Usp proteins were incubated with the EcR consensus probe hspEcRBE for 20 min in the presence of dronc promoter fragments. 400 bp PCR fragments spanning the dronc promoter region from 2.8 to 1.1 kb were gel purified and 400 ng was used in each reaction. Positive control ( hsp EcRBE) was used at 40 ng (equimolar). Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–UsP complex is indicated. (B) EMSA experiment was performed as in A except PCR fragments used as competitors spanned the regions from 1.42 to 1.0 kb. hspEcRBE was used as the probe. (C) EMSA was performed as in A. Negative control competitor (D4) corresponds to the dronc promoter region between 67 to 7 bp upstream of the transcription start site. Oligonucleotide competitors correspond to the dronc promoter region 1.44 to 1.2 kb upstream of the transcription start site. Hsp EcRBE was used as the probe.
Figure Legend Snippet: dronc upstream promoter harbors a putative EcRBE. (A) In vitro translated EcR and Usp proteins were incubated with the EcR consensus probe hspEcRBE for 20 min in the presence of dronc promoter fragments. 400 bp PCR fragments spanning the dronc promoter region from 2.8 to 1.1 kb were gel purified and 400 ng was used in each reaction. Positive control ( hsp EcRBE) was used at 40 ng (equimolar). Complexes were resolved on an acrylamide/TBE gel, dried on 3 MM Whatmann paper, and exposed to Kodak film overnight. EcR–UsP complex is indicated. (B) EMSA experiment was performed as in A except PCR fragments used as competitors spanned the regions from 1.42 to 1.0 kb. hspEcRBE was used as the probe. (C) EMSA was performed as in A. Negative control competitor (D4) corresponds to the dronc promoter region between 67 to 7 bp upstream of the transcription start site. Oligonucleotide competitors correspond to the dronc promoter region 1.44 to 1.2 kb upstream of the transcription start site. Hsp EcRBE was used as the probe.

Techniques Used: In Vitro, Incubation, Polymerase Chain Reaction, Purification, Positive Control, Negative Control

Salivary glands and midgut express EcR-B1, which binds to dronc promoter. (A) Salivary glands and midgut were dissected from animals at −24, 2, or 12 h relative to puparium formation. RNA was analyzed by RT-PCR to detect EcR isoform expression. Rp49 was used as a control. (B) 9 μg of nuclear extracts prepared from various staged animals were incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min. Complexes were analyzed as in Figs. 4 and 5 . EcR–Usp complex and supershift (ss) are indicated. Developmental stages are shown as hours relative to puparium formation. These stages represent early (0–4 h), mid (5–6 h), and late (9–11 h) prepupae and early (11–12 h) pupae. (C) EMSA was performed as in B in the presence of dronc EcRBE cold competitor, 2 μl of EcR common antibody, EcR-B1, or EcR-A. EcR–Usp (EcR) and supershifted EcR–Usp (ss) complexes are indicated. (D) EMSA was performed as in C with 6 μg on nuclear extract from salivary glands or midguts from 12 h (salivary gland) and 2 h (midgut) staged prepupae. EcR–Usp and supershifted (ss) complexes are shown. 40 ng of cold competitor ( dronc EcRBE) was also added where indicated.
Figure Legend Snippet: Salivary glands and midgut express EcR-B1, which binds to dronc promoter. (A) Salivary glands and midgut were dissected from animals at −24, 2, or 12 h relative to puparium formation. RNA was analyzed by RT-PCR to detect EcR isoform expression. Rp49 was used as a control. (B) 9 μg of nuclear extracts prepared from various staged animals were incubated with the dronc EcRBE or the EcRBE mutant probe for 20 min. Complexes were analyzed as in Figs. 4 and 5 . EcR–Usp complex and supershift (ss) are indicated. Developmental stages are shown as hours relative to puparium formation. These stages represent early (0–4 h), mid (5–6 h), and late (9–11 h) prepupae and early (11–12 h) pupae. (C) EMSA was performed as in B in the presence of dronc EcRBE cold competitor, 2 μl of EcR common antibody, EcR-B1, or EcR-A. EcR–Usp (EcR) and supershifted EcR–Usp (ss) complexes are indicated. (D) EMSA was performed as in C with 6 μg on nuclear extract from salivary glands or midguts from 12 h (salivary gland) and 2 h (midgut) staged prepupae. EcR–Usp and supershifted (ss) complexes are shown. 40 ng of cold competitor ( dronc EcRBE) was also added where indicated.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Mutagenesis

22) Product Images from "The Promoter of the pri-miR-375 Gene Directs Expression Selectively to the Endocrine Pancreas"

Article Title: The Promoter of the pri-miR-375 Gene Directs Expression Selectively to the Endocrine Pancreas

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005033

Mapping of transcription start site of miR-375 gene by 5′-RACE. A. DNase-treated total RNA from HIT cells transfected with the pGL3-375a construct was reverse transcribed, using a primer complementary to the luciferase sequence. The cDNA was poly-(dG) tailed and amplified by PCR using a poly-C primer and a nested primer complementary to block 4 (lane 1). Lane 2, no RT control; lane 3, no template control. Marker sizes are indicated. The location of the band in lane 1 is indicated by an arrow. B. Alignment of miR-375 upstream sequences (from −96 to +244, relative to transcription start site) of mouse, rat and human. The primer used for PCR amplification is located at the middle of block 4. The TATA sequence is marked by a box. The large arrow-head indicates the major transcription start site. Small arrow-heads indicate the start site of shorter species detected by 5′-RACE. C. Representation of the pGL3-375a construct. Conserved regions 1–4 are indicated. The luciferase gene is indicated as an unmarked open box. The arrow indicates the transcription start site revealed by 5′-RACE analysis. Numbers above the bar indicate location relative to the transcription start site.
Figure Legend Snippet: Mapping of transcription start site of miR-375 gene by 5′-RACE. A. DNase-treated total RNA from HIT cells transfected with the pGL3-375a construct was reverse transcribed, using a primer complementary to the luciferase sequence. The cDNA was poly-(dG) tailed and amplified by PCR using a poly-C primer and a nested primer complementary to block 4 (lane 1). Lane 2, no RT control; lane 3, no template control. Marker sizes are indicated. The location of the band in lane 1 is indicated by an arrow. B. Alignment of miR-375 upstream sequences (from −96 to +244, relative to transcription start site) of mouse, rat and human. The primer used for PCR amplification is located at the middle of block 4. The TATA sequence is marked by a box. The large arrow-head indicates the major transcription start site. Small arrow-heads indicate the start site of shorter species detected by 5′-RACE. C. Representation of the pGL3-375a construct. Conserved regions 1–4 are indicated. The luciferase gene is indicated as an unmarked open box. The arrow indicates the transcription start site revealed by 5′-RACE analysis. Numbers above the bar indicate location relative to the transcription start site.

Techniques Used: Transfection, Construct, Luciferase, Sequencing, Amplification, Polymerase Chain Reaction, Blocking Assay, Marker

23) Product Images from "A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance"

Article Title: A single-base substitution within an intronic repetitive element causes dominant retinitis pigmentosa with reduced penetrance

Journal: Human mutation

doi: 10.1002/humu.21071

PRPF31 mRNA isoforms in LCLs from members of family #1562 (A) A representative image of RT-PCR products, run on a 1.5% agarose gel, derived from the exon 13-exon 14 mRNA segment from 2 asymptomatic (III-9, III-11) and 1 affected (IV-16) individuals from family #1562, as well as from 1 CEPH individual (Ctrl). “M” is 100 bp ladder. Length and names of PCR products are indicated on the right side of the gel. (B) Schematic representation of the 3 mRNA isoforms from which PCR products are derived. Numbered rectangles and bold lines represent exons and introns of PRPF31 , respectively. Hatched rectangles designate parts of intron 13 that were retained (starts and ends are indicated by numbers). Numbering of stop codons for each mRNA isoform is based on the wild-type sequence. Depicted exons and introns are not to scale.
Figure Legend Snippet: PRPF31 mRNA isoforms in LCLs from members of family #1562 (A) A representative image of RT-PCR products, run on a 1.5% agarose gel, derived from the exon 13-exon 14 mRNA segment from 2 asymptomatic (III-9, III-11) and 1 affected (IV-16) individuals from family #1562, as well as from 1 CEPH individual (Ctrl). “M” is 100 bp ladder. Length and names of PCR products are indicated on the right side of the gel. (B) Schematic representation of the 3 mRNA isoforms from which PCR products are derived. Numbered rectangles and bold lines represent exons and introns of PRPF31 , respectively. Hatched rectangles designate parts of intron 13 that were retained (starts and ends are indicated by numbers). Numbering of stop codons for each mRNA isoform is based on the wild-type sequence. Depicted exons and introns are not to scale.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Derivative Assay, Polymerase Chain Reaction, Sequencing

24) Product Images from "Transcription Factor PrtT Controls Expression of Multiple Secreted Proteases in the Human Pathogenic Mold Aspergillus fumigatus "

Article Title: Transcription Factor PrtT Controls Expression of Multiple Secreted Proteases in the Human Pathogenic Mold Aspergillus fumigatus

Journal: Infection and Immunity

doi: 10.1128/IAI.00426-09

The expression of six secreted A. fumigatus proteases is markedly reduced by deletion of prtT . Dormant conidia were incubated for 12, 24, 48, and 72 h in liquid SM medium at 37°C, after which total RNA was isolated and used in RT-PCR. Expression
Figure Legend Snippet: The expression of six secreted A. fumigatus proteases is markedly reduced by deletion of prtT . Dormant conidia were incubated for 12, 24, 48, and 72 h in liquid SM medium at 37°C, after which total RNA was isolated and used in RT-PCR. Expression

Techniques Used: Expressing, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction

25) Product Images from "Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator"

Article Title: Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001255

DNAJC14 confers resistance to YFV-induced cell death. (A) Photographs 7 d after YFV challenge (moi = 1) of SW13 cells transduced with Round 3 of the selected lentiviral cDNA constructs compared to cells transduced with V1-GFP vector control. (B) The cells transduced with the Round 3 lentivirus pool and surviving YFV infection (Rd 3) were expanded and reinfected with YFV at the indicated moi. Crystal violet staining was performed 3 d later. Cells transduced with vector alone serve as a control (V1-GFP). (C) DNA was isolated from naïve SW13 or Round 3 (Rd 3) cells, and the lentiviral insert amplified by PCR. The major band was identified as encoding a truncated hamster DNAJC14. Sizes of the DNA markers (kb) are indicated to the left. (D) A schematic of human DNAJC14 is shown, with the putative transmembrane (TM) domains (gray), J domain (red) with conserved HPD sequence, zinc finger motifs (blue) and Jiv90 domain (orange) indicated. A schematic of the isolated hamster clone, showing homology to amino acids 305 to 702 of human DNAJC14, is shown below.
Figure Legend Snippet: DNAJC14 confers resistance to YFV-induced cell death. (A) Photographs 7 d after YFV challenge (moi = 1) of SW13 cells transduced with Round 3 of the selected lentiviral cDNA constructs compared to cells transduced with V1-GFP vector control. (B) The cells transduced with the Round 3 lentivirus pool and surviving YFV infection (Rd 3) were expanded and reinfected with YFV at the indicated moi. Crystal violet staining was performed 3 d later. Cells transduced with vector alone serve as a control (V1-GFP). (C) DNA was isolated from naïve SW13 or Round 3 (Rd 3) cells, and the lentiviral insert amplified by PCR. The major band was identified as encoding a truncated hamster DNAJC14. Sizes of the DNA markers (kb) are indicated to the left. (D) A schematic of human DNAJC14 is shown, with the putative transmembrane (TM) domains (gray), J domain (red) with conserved HPD sequence, zinc finger motifs (blue) and Jiv90 domain (orange) indicated. A schematic of the isolated hamster clone, showing homology to amino acids 305 to 702 of human DNAJC14, is shown below.

Techniques Used: Transduction, Construct, Plasmid Preparation, Infection, Staining, Isolation, Amplification, Polymerase Chain Reaction, Sequencing

26) Product Images from "Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells *"

Article Title: Transcriptional Regulation of Tlr11 Gene Expression in Epithelial Cells *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.050757

Expression of murine Tlr11 gene in epithelial cells and determination of the Tlr11 transcription start site by 5′-RACE PCR. A, expression of Tlr11 in murine epithelial cells. Total RNA from mouse embryo fibroblast ( MEF ), macrophage Raw 264.7,
Figure Legend Snippet: Expression of murine Tlr11 gene in epithelial cells and determination of the Tlr11 transcription start site by 5′-RACE PCR. A, expression of Tlr11 in murine epithelial cells. Total RNA from mouse embryo fibroblast ( MEF ), macrophage Raw 264.7,

Techniques Used: Expressing, Polymerase Chain Reaction

Effects of the knocked down expression of ESE-1, ESE-3, and IRF8 on Tlr11 endogenous expression and promoter activity. A, real time RT-PCR analysis of Tlr11 expression in knocked down cells. Total RNA extracted from ESE-1, ESE-3, and IRF8 knocked down
Figure Legend Snippet: Effects of the knocked down expression of ESE-1, ESE-3, and IRF8 on Tlr11 endogenous expression and promoter activity. A, real time RT-PCR analysis of Tlr11 expression in knocked down cells. Total RNA extracted from ESE-1, ESE-3, and IRF8 knocked down

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

27) Product Images from "Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway"

Article Title: Expression of Borrelia burgdorferi OspC and DbpA is controlled by a RpoN-RpoS regulatory pathway

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

doi: 10.1073/pnas.231442498

PCR analysis of rpoN and rpoS mutants. ( a ) Schematic of PCR primer pairs (short arrows). ( b and c ) Agarose gel patterns of amplicons for wild type (lanes 2–4), ( b ) a rpoN mutant, or ( c ) a rpoS mutant (lanes 5–7). Lanes 1 contain DNA markers of ΦX174/ Hae III. Gene disruption by ermC results in an increased size of the amplicons (compare lanes 2 and 5). A combination of ermC -specific and flanking primers yielded only products for the mutants (lanes 6 and 7), but not for 297 (lanes 3 and 4). Lanes 3 and 6: priAH102 (arrow 102) in combination with ( b ) priAH59 (arrow 59) or ( c ) priAH27 (arrow 27). Lanes 4 and 7: priAH104 (arrow 104) in combination with ( b ) priAH60 (arrow 60) or ( c ) priAH131 (arrow 131).
Figure Legend Snippet: PCR analysis of rpoN and rpoS mutants. ( a ) Schematic of PCR primer pairs (short arrows). ( b and c ) Agarose gel patterns of amplicons for wild type (lanes 2–4), ( b ) a rpoN mutant, or ( c ) a rpoS mutant (lanes 5–7). Lanes 1 contain DNA markers of ΦX174/ Hae III. Gene disruption by ermC results in an increased size of the amplicons (compare lanes 2 and 5). A combination of ermC -specific and flanking primers yielded only products for the mutants (lanes 6 and 7), but not for 297 (lanes 3 and 4). Lanes 3 and 6: priAH102 (arrow 102) in combination with ( b ) priAH59 (arrow 59) or ( c ) priAH27 (arrow 27). Lanes 4 and 7: priAH104 (arrow 104) in combination with ( b ) priAH60 (arrow 60) or ( c ) priAH131 (arrow 131).

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

28) Product Images from "Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs"

Article Title: Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs

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

doi: 10.1073/pnas.211399398

PCR analysis of the genomic DNA extracted from HMEC-1 cells nontransduced or transduced with various retroviral vectors. Different combinations of primers were used to amplify HHO-1, neo r , or chimeric vector/HHO-1 DNA fragments. P1 and P2, primers for amplifying LXSN vector sequence; P3 and P4, primers for neo r gene; P5 and P6, primers for HHO-1 DNA fragment. The combination of P1 and P5 for HHO-1-AS-transduced HMEC-1 cells (HMEC/HOP-HHO-1-AS) detected a 2,441-bp signal that contains partial vector sequence, HOP, and a partial HHO-1-AS DNA fragment. Similarly, a 2,260-bp PCR product containing partial vector sequence, HOP, and a partial HHO-1 gene fragment was amplified from HHO-1-transduced cells (HMEC/HOP-HHO-1) when primers P1 and P6 were used. The primers P1 and P2 amplified a 1,661-bp PCR product from control vector (LSN-HOP)-transduced HMEC-1 cells (HMEC/HOP). The 313-bp neo r DNA fragments were detected in all three kinds of cells mentioned above by using primers P3 and P4.
Figure Legend Snippet: PCR analysis of the genomic DNA extracted from HMEC-1 cells nontransduced or transduced with various retroviral vectors. Different combinations of primers were used to amplify HHO-1, neo r , or chimeric vector/HHO-1 DNA fragments. P1 and P2, primers for amplifying LXSN vector sequence; P3 and P4, primers for neo r gene; P5 and P6, primers for HHO-1 DNA fragment. The combination of P1 and P5 for HHO-1-AS-transduced HMEC-1 cells (HMEC/HOP-HHO-1-AS) detected a 2,441-bp signal that contains partial vector sequence, HOP, and a partial HHO-1-AS DNA fragment. Similarly, a 2,260-bp PCR product containing partial vector sequence, HOP, and a partial HHO-1 gene fragment was amplified from HHO-1-transduced cells (HMEC/HOP-HHO-1) when primers P1 and P6 were used. The primers P1 and P2 amplified a 1,661-bp PCR product from control vector (LSN-HOP)-transduced HMEC-1 cells (HMEC/HOP). The 313-bp neo r DNA fragments were detected in all three kinds of cells mentioned above by using primers P3 and P4.

Techniques Used: Polymerase Chain Reaction, Transduction, Plasmid Preparation, Sequencing, Amplification

( A ) Northern blot analysis of RNA obtained from PA317 and NIH 3T3 cells nontransduced or transduced with retroviral vector LSN-HHO-1 or LXSN. Lanes 1 and 2, PA317/HHO-1 cells; lane 3, control PA317 retroviral packaging cells; lane 4, PA317/LXSN cells; lane 5, NIH 3T3/LXSN cells; lanes 6 and 7, NIH 3T3/HHO-1 cells; lane 8, control NIH 3T3 cells. ( B ) Reverse transcription (RT)-PCR analysis of PT67 retroviral packaging cells transduced with different retroviral vectors. Lanes 1 and 6, PT67 cells; lanes 2 and 7, PT67/LXSN cells; lanes 3 and 8, PT67/LSN-HOP cells; lanes 4 and 9, PT67/LSN-HOP-HHO-1 cells; Lanes 5 and 10, PT67/LSN-HOP-HHO-1-AS cells. m, Hin dIII-digested λ DNA marker; M, 100-bp DNA ruler; neo r , RT-PCR products of neomycin-resistance gene; HO-1, human HO-1 RT-PCR products. ( C ) Detection of human (h)HO-1 and G3PDH transcripts by Northern blot analysis in RLMV cells transduced with retroviral vectors LSN-HHO-1 or LXSN. Lanes 1, 2, and 5, RLMV cells transduced with LSN-HHO-1; lane 3, control RLMV cells; lane 4, RLMV cells transduced with LXSN.
Figure Legend Snippet: ( A ) Northern blot analysis of RNA obtained from PA317 and NIH 3T3 cells nontransduced or transduced with retroviral vector LSN-HHO-1 or LXSN. Lanes 1 and 2, PA317/HHO-1 cells; lane 3, control PA317 retroviral packaging cells; lane 4, PA317/LXSN cells; lane 5, NIH 3T3/LXSN cells; lanes 6 and 7, NIH 3T3/HHO-1 cells; lane 8, control NIH 3T3 cells. ( B ) Reverse transcription (RT)-PCR analysis of PT67 retroviral packaging cells transduced with different retroviral vectors. Lanes 1 and 6, PT67 cells; lanes 2 and 7, PT67/LXSN cells; lanes 3 and 8, PT67/LSN-HOP cells; lanes 4 and 9, PT67/LSN-HOP-HHO-1 cells; Lanes 5 and 10, PT67/LSN-HOP-HHO-1-AS cells. m, Hin dIII-digested λ DNA marker; M, 100-bp DNA ruler; neo r , RT-PCR products of neomycin-resistance gene; HO-1, human HO-1 RT-PCR products. ( C ) Detection of human (h)HO-1 and G3PDH transcripts by Northern blot analysis in RLMV cells transduced with retroviral vectors LSN-HHO-1 or LXSN. Lanes 1, 2, and 5, RLMV cells transduced with LSN-HHO-1; lane 3, control RLMV cells; lane 4, RLMV cells transduced with LXSN.

Techniques Used: Northern Blot, Transduction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Marker

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

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

Journal:

doi: 10.1128/IAI.00288-07

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

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Western Blot

30) Product Images from "Degradation of Pheromone and Plant Volatile Components by a Same Odorant-Degrading Enzyme in the Cotton Leafworm, Spodoptera littoralis"

Article Title: Degradation of Pheromone and Plant Volatile Components by a Same Odorant-Degrading Enzyme in the Cotton Leafworm, Spodoptera littoralis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0029147

Comparison of SlCXE7 expression in S. littoralis tissues. A ) Coomassie staining after SDS-PAGE of adult tissues. B ) Western-blot with anti-SlCXE7 specific antibody on the same tissues. C ) Non-quantitative RT-PCR analysis on tissues from last instar larvae. M: male; f: female; ant, antennae; prob, proboscis; abd, abdomen.
Figure Legend Snippet: Comparison of SlCXE7 expression in S. littoralis tissues. A ) Coomassie staining after SDS-PAGE of adult tissues. B ) Western-blot with anti-SlCXE7 specific antibody on the same tissues. C ) Non-quantitative RT-PCR analysis on tissues from last instar larvae. M: male; f: female; ant, antennae; prob, proboscis; abd, abdomen.

Techniques Used: Expressing, Staining, SDS Page, Western Blot, Quantitative RT-PCR

31) Product Images from "Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment"

Article Title: Grsf1-Induced Translation of the SNARE Protein Use1 Is Required for Expansion of the Erythroid Compartment

Journal: PLoS ONE

doi: 10.1371/journal.pone.0104631

Grsf1 controls translation of Use1. ( A ) Luciferase reporter constructs harboring the Use1 wt 5′UTR (WT, black bars), or a 5′UTR lacking the AGGGCGGA repeat (open bars), were transfected in NIH3T3 cells together with increasing amounts of a Grsf1 expression plasmid. The total amount of transfected DNA was kept constant by adding additional backbone plasmid DNA (EV, pcDNA3.1). Luciferase activity is corrected for mRNA expression and given as fold-change (fc) compared to WT reporter in absence of Grsf1 overexpression. Error bars indicate standard deviation of 3 experiments. ( B ) I/11cells were transduced with the lentiviral constructs containing two different shRNAs matching Grsf1 (indicated a and b) or control shRNA (Scr). The efficiency of Grsf1 transcript knock down was determined using RT-PCR. The expression of Grsf1 in parental cells is set to 1. Error bars indicate standard deviation of 3 experiments. ( C ) The protein lysates from transduced I/11 cells (representative experiment from B) were tested in WB with anti-actin (42 kDa) and anti-Use1 (30.5 kDa) antibody.
Figure Legend Snippet: Grsf1 controls translation of Use1. ( A ) Luciferase reporter constructs harboring the Use1 wt 5′UTR (WT, black bars), or a 5′UTR lacking the AGGGCGGA repeat (open bars), were transfected in NIH3T3 cells together with increasing amounts of a Grsf1 expression plasmid. The total amount of transfected DNA was kept constant by adding additional backbone plasmid DNA (EV, pcDNA3.1). Luciferase activity is corrected for mRNA expression and given as fold-change (fc) compared to WT reporter in absence of Grsf1 overexpression. Error bars indicate standard deviation of 3 experiments. ( B ) I/11cells were transduced with the lentiviral constructs containing two different shRNAs matching Grsf1 (indicated a and b) or control shRNA (Scr). The efficiency of Grsf1 transcript knock down was determined using RT-PCR. The expression of Grsf1 in parental cells is set to 1. Error bars indicate standard deviation of 3 experiments. ( C ) The protein lysates from transduced I/11 cells (representative experiment from B) were tested in WB with anti-actin (42 kDa) and anti-Use1 (30.5 kDa) antibody.

Techniques Used: Luciferase, Construct, Transfection, Expressing, Plasmid Preparation, Activity Assay, Over Expression, Standard Deviation, Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot

The unspliced transcript is preferentially translated. ( A ) Polysome profile of I/ll cells. Cell lysate was centrifuged on a 7–46% sucrose gradient and the distribution of RNA was measured by absorption at 254 nm. The free mRNA, ribosomal subunits and polysomes are indicated. ( B ) Real time, reverse transcriptase PCR on polysome bound (pb) and subpolysomal (sub) mRNA using a reverse primer downstream the Use1 AUG start codon and forward primers upstream of, and within the intron to amplify the spliced (black bars) and unspliced (white bars) RNA, respectively. The percentage polysome recruitment was calculated for both transcripts. ( C ) The spliced mRNA variant encodes a longer protein variant. The top nucleotide sequence represents the unspliced transcript. At −154 and +3 the AG nucleotides that are part of the splice sites are shown in capitals. The lower nucleotide sequence represents the spliced transcript. A start codon at nucleotide −108, compliant with the Kazak consensus, appears to be in frame with the main ORF in the spliced transcript, whereas it is followed by stop codon in the intron in the unspliced transcript. Predicted protein sequences are shown above the nucleotide sequences. Slashes indicate gaps in the sequence as only important parts of the sequence are shown. ( D ) Protein expression in cultured I/11 erythroblasts and in primary liver cells (C57/B6). Western blot probed with anti-Use1, and with anti-Stat3 as loading control. Use 1 isoforms are indicated.
Figure Legend Snippet: The unspliced transcript is preferentially translated. ( A ) Polysome profile of I/ll cells. Cell lysate was centrifuged on a 7–46% sucrose gradient and the distribution of RNA was measured by absorption at 254 nm. The free mRNA, ribosomal subunits and polysomes are indicated. ( B ) Real time, reverse transcriptase PCR on polysome bound (pb) and subpolysomal (sub) mRNA using a reverse primer downstream the Use1 AUG start codon and forward primers upstream of, and within the intron to amplify the spliced (black bars) and unspliced (white bars) RNA, respectively. The percentage polysome recruitment was calculated for both transcripts. ( C ) The spliced mRNA variant encodes a longer protein variant. The top nucleotide sequence represents the unspliced transcript. At −154 and +3 the AG nucleotides that are part of the splice sites are shown in capitals. The lower nucleotide sequence represents the spliced transcript. A start codon at nucleotide −108, compliant with the Kazak consensus, appears to be in frame with the main ORF in the spliced transcript, whereas it is followed by stop codon in the intron in the unspliced transcript. Predicted protein sequences are shown above the nucleotide sequences. Slashes indicate gaps in the sequence as only important parts of the sequence are shown. ( D ) Protein expression in cultured I/11 erythroblasts and in primary liver cells (C57/B6). Western blot probed with anti-Use1, and with anti-Stat3 as loading control. Use 1 isoforms are indicated.

Techniques Used: Polymerase Chain Reaction, Variant Assay, Sequencing, Expressing, Cell Culture, Western Blot

Reduced expression of Use1 or Grsf1 abrogates expansion of erythroblasts. Erythroblasts (I/11 cell line) were transduced with lentiviral shRNA vectors repressing expression of Use1 (2 distinct sequences, indicated a and b) or Grsf1 (2 distinct sequences, indicated a and b). Panels show representative data from 1 of 4 replicates. ( A ) Use1 RNA expression was controlled by RT-PCR 4 days following transfection. For Grsf1 knockdown see figure 4B ) ( B ) Cytospins of the cultured cells made 4 days post transduction showed mainly blasts when expressing control shRNA (Scr). Expression of shRNAs matching Use1 or Grsf1 increased the number of more mature hemoglobinised cells (Dark staining, white arrows) and pycnotic cells (grey arrows). A quantification of cell types, based on counting > 300 cells, is shown above the cytospin as pie-diagram (black, blasts; white, maturing red cells; grey, pycnotic cells). ( C ) shRNA treated cells were counted daily, kept at 2×10 6 /ml and cumulative cell numbers were calculated. ( D ) At the start and 96 hours following induction of differentiation the hemoglobin concentration was measured and calculated as Hb/cell volume in arbitrary units (A.U.). ( E ) At 96 hours the ratio of life/dead cells was determined by staining with propidium iodide (PI). Cells positive for PI were measured by flowcytometry (LSRII, BD).
Figure Legend Snippet: Reduced expression of Use1 or Grsf1 abrogates expansion of erythroblasts. Erythroblasts (I/11 cell line) were transduced with lentiviral shRNA vectors repressing expression of Use1 (2 distinct sequences, indicated a and b) or Grsf1 (2 distinct sequences, indicated a and b). Panels show representative data from 1 of 4 replicates. ( A ) Use1 RNA expression was controlled by RT-PCR 4 days following transfection. For Grsf1 knockdown see figure 4B ) ( B ) Cytospins of the cultured cells made 4 days post transduction showed mainly blasts when expressing control shRNA (Scr). Expression of shRNAs matching Use1 or Grsf1 increased the number of more mature hemoglobinised cells (Dark staining, white arrows) and pycnotic cells (grey arrows). A quantification of cell types, based on counting > 300 cells, is shown above the cytospin as pie-diagram (black, blasts; white, maturing red cells; grey, pycnotic cells). ( C ) shRNA treated cells were counted daily, kept at 2×10 6 /ml and cumulative cell numbers were calculated. ( D ) At the start and 96 hours following induction of differentiation the hemoglobin concentration was measured and calculated as Hb/cell volume in arbitrary units (A.U.). ( E ) At 96 hours the ratio of life/dead cells was determined by staining with propidium iodide (PI). Cells positive for PI were measured by flowcytometry (LSRII, BD).

Techniques Used: Expressing, Transduction, shRNA, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Transfection, Cell Culture, Staining, Concentration Assay

32) Product Images from "A missense mutation (Q279R) in the Fumarylacetoacetate Hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation"

Article Title: A missense mutation (Q279R) in the Fumarylacetoacetate Hydrolase gene, responsible for hereditary tyrosinemia, acts as a splicing mutation

Journal: BMC Genetics

doi: 10.1186/1471-2156-2-9

Mutation analysis in different liver regions. DNA was extracted from different liver regions and amplified by PCR. PCR products were digested with either Alu I to detect IVS6-1g- > t or with Msp I to detect Q279R. For IVS6-1 g- > t, the same heterozygous pattern is seen in both the reverted nodule (NT), tumor section (T) and fibroblast DNA (F), showing 3 bands, one at 156-, another at 104- and the last at 75-bp. The control (wt/wt) shows two bands, one at 156- and the other at 75-bp, indicating the absence of IVS6-1g- > t (M: molecular weight marker, 100- and 200-bp). For Q279R both the 78- and 58-bp bands are seen in the tumor section (T) and fibroblast DNA (F) indicating an heterozygous genotype while only the 78-bp wild-type band is seen in the control (wt/wt). In the region suspected of reversion (NT), a strong 78-bp wild-type band is seen with a weak 58-bp mutated band (M: molecular weight marker, 100-bp).
Figure Legend Snippet: Mutation analysis in different liver regions. DNA was extracted from different liver regions and amplified by PCR. PCR products were digested with either Alu I to detect IVS6-1g- > t or with Msp I to detect Q279R. For IVS6-1 g- > t, the same heterozygous pattern is seen in both the reverted nodule (NT), tumor section (T) and fibroblast DNA (F), showing 3 bands, one at 156-, another at 104- and the last at 75-bp. The control (wt/wt) shows two bands, one at 156- and the other at 75-bp, indicating the absence of IVS6-1g- > t (M: molecular weight marker, 100- and 200-bp). For Q279R both the 78- and 58-bp bands are seen in the tumor section (T) and fibroblast DNA (F) indicating an heterozygous genotype while only the 78-bp wild-type band is seen in the control (wt/wt). In the region suspected of reversion (NT), a strong 78-bp wild-type band is seen with a weak 58-bp mutated band (M: molecular weight marker, 100-bp).

Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Molecular Weight, Marker

Analysis of the splicing pattern obtained with the minigenes. A- The splicing K7 consists of exon 1 of β-globin and its downstream intronic sequences joined to β-globin exon 3 and its upstream intronic sequences. Exon 9, with or without the Q279R mutation was inserted in K7 at the intronic junction. HeLa cells were transiently transfected with both constructs, the wild-type Q279Q-K7 and Q279R-containing Q279R-K7. After 24 hours, cells were harvested and the splicing pattern of each minigene was examined by RT-PCR analysis of the transcripts. Exons are represented by boxes and introns by lines. The primers used for RT-PCR are indicated at each end of the splicing K7. B- Total RNA extracted from transfected HeLa cells was amplified with HG1S and HG3AS. Plasmidic DNA Q279Q-K7 and Q279R-K7 (pDNA) were also amplified as a control. The band obtained for Q279Q-K7 transfected cells (RT+) is of expected size, in contrast to the band obtained in the case of Q279R-K7 (RT+) transfected cells, which is of lower molecular weight. RT - serves as a negative control: the reverse transcription reaction was performed without any enzyme. In the two RT - fractions, the amplification of about 900-bp is due to plasmidic DNA contamination.
Figure Legend Snippet: Analysis of the splicing pattern obtained with the minigenes. A- The splicing K7 consists of exon 1 of β-globin and its downstream intronic sequences joined to β-globin exon 3 and its upstream intronic sequences. Exon 9, with or without the Q279R mutation was inserted in K7 at the intronic junction. HeLa cells were transiently transfected with both constructs, the wild-type Q279Q-K7 and Q279R-containing Q279R-K7. After 24 hours, cells were harvested and the splicing pattern of each minigene was examined by RT-PCR analysis of the transcripts. Exons are represented by boxes and introns by lines. The primers used for RT-PCR are indicated at each end of the splicing K7. B- Total RNA extracted from transfected HeLa cells was amplified with HG1S and HG3AS. Plasmidic DNA Q279Q-K7 and Q279R-K7 (pDNA) were also amplified as a control. The band obtained for Q279Q-K7 transfected cells (RT+) is of expected size, in contrast to the band obtained in the case of Q279R-K7 (RT+) transfected cells, which is of lower molecular weight. RT - serves as a negative control: the reverse transcription reaction was performed without any enzyme. In the two RT - fractions, the amplification of about 900-bp is due to plasmidic DNA contamination.

Techniques Used: Mutagenesis, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Amplification, Molecular Weight, Negative Control

33) Product Images from "The Myostatin Gene Is a Downstream Target Gene of Basic Helix-Loop-Helix Transcription Factor MyoD"

Article Title: The Myostatin Gene Is a Downstream Target Gene of Basic Helix-Loop-Helix Transcription Factor MyoD

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.22.20.7066-7082.2002

Mutational analysis of E-box motifs in the proximal cluster of the bovine myostatin promoter. Mutations in the individual E-box motifs (E3M, E4M, or E6M) or combinations thereof (E3M+E4M and E3M+E4M+E6M) were generated by PCR, and the promoter fragments containing the mutations were cloned into the reporter vector pGL3-Basic. The percentage of luciferase activity was derived by comparing the luciferase activity of each mutant construct with that of the 1.6 construct. The activity of the 1.6 construct is represented as 100%. Bars indicate means ± standard deviations for six replicates. ∗, P
Figure Legend Snippet: Mutational analysis of E-box motifs in the proximal cluster of the bovine myostatin promoter. Mutations in the individual E-box motifs (E3M, E4M, or E6M) or combinations thereof (E3M+E4M and E3M+E4M+E6M) were generated by PCR, and the promoter fragments containing the mutations were cloned into the reporter vector pGL3-Basic. The percentage of luciferase activity was derived by comparing the luciferase activity of each mutant construct with that of the 1.6 construct. The activity of the 1.6 construct is represented as 100%. Bars indicate means ± standard deviations for six replicates. ∗, P

Techniques Used: Generated, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Derivative Assay, Mutagenesis, Construct

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

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

Journal: Genes, chromosomes & cancer

doi: 10.1002/gcc.20731

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

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

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

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Sequencing

35) Product Images from "Next Generation Sequencing and Animal Models Reveal SLC9A3R1 as a New Gene Involved in Human Age-Related Hearing Loss"

Article Title: Next Generation Sequencing and Animal Models Reveal SLC9A3R1 as a New Gene Involved in Human Age-Related Hearing Loss

Journal: Frontiers in Genetics

doi: 10.3389/fgene.2019.00142

CRISPR-Cas9 strategy for the generation of the R180Q- slc9a3r1 K/I model. (A) Comparison of human and zebrafish SLC9A3R1 amino acid sequence. Purple rectangle outlines R180 conservation among human and zebrafish. (B) Scheme displaying the nucleotide and amino acid translated sequence present at the target genome (below) and the ssOligo (above) used for modifying the target sequence. Below, green arrow in genome sequence outlines the sgRNA site. Above, red squares in ssOligo outline homologous recombination right and left arms. (C) Scheme displaying the PCR strategy used to identify potential mutants. (D) Example of R180Q-Slc9a3r1 mutant identified through the comparison of Sanger sequences obtained from a wild type (above) and a heterozygous individual (below). Both sequences display nucleotide and translated amino acid sequence. Above, targeted amino acid R180 is displayed in green. Below, modified amin oacid Q180 is displayed in red and modified nucleotides are displayed in red and lower case.
Figure Legend Snippet: CRISPR-Cas9 strategy for the generation of the R180Q- slc9a3r1 K/I model. (A) Comparison of human and zebrafish SLC9A3R1 amino acid sequence. Purple rectangle outlines R180 conservation among human and zebrafish. (B) Scheme displaying the nucleotide and amino acid translated sequence present at the target genome (below) and the ssOligo (above) used for modifying the target sequence. Below, green arrow in genome sequence outlines the sgRNA site. Above, red squares in ssOligo outline homologous recombination right and left arms. (C) Scheme displaying the PCR strategy used to identify potential mutants. (D) Example of R180Q-Slc9a3r1 mutant identified through the comparison of Sanger sequences obtained from a wild type (above) and a heterozygous individual (below). Both sequences display nucleotide and translated amino acid sequence. Above, targeted amino acid R180 is displayed in green. Below, modified amin oacid Q180 is displayed in red and modified nucleotides are displayed in red and lower case.

Techniques Used: CRISPR, Sequencing, Homologous Recombination, Polymerase Chain Reaction, Mutagenesis, Modification

36) Product Images from "Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat"

Article Title: Investigations of barley stripe mosaic virus as a gene silencing vector in barley roots and in Brachypodium distachyon and oat

Journal: Plant Methods

doi: 10.1186/1746-4811-6-26

Silencing of BdPDS in B. distachyon leaves A Leaves of B. distachyon infected with BSMV-GFP 375 or BSMV-BdPDS. B BdPDS RNA expression levels in experiments 2 and 3 (Table 1) determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-BdPDS (black bars) or BSMV-GFP 375 (white bars). From left to right, the bars represent 16, 13, 6 and 6 samples, respectively. Error bars denote standard deviations. AU - Arbitrary units. Significantly differences: * ( p
Figure Legend Snippet: Silencing of BdPDS in B. distachyon leaves A Leaves of B. distachyon infected with BSMV-GFP 375 or BSMV-BdPDS. B BdPDS RNA expression levels in experiments 2 and 3 (Table 1) determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-BdPDS (black bars) or BSMV-GFP 375 (white bars). From left to right, the bars represent 16, 13, 6 and 6 samples, respectively. Error bars denote standard deviations. AU - Arbitrary units. Significantly differences: * ( p

Techniques Used: Infection, RNA Expression, Quantitative RT-PCR

Silencing of HvIPS1 in barley roots and shoots . A HvIPS1 RNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants inoculated with BSMV-IPS1 (black bars) or BSMV-GFP 250 (white bars). Plants were grown in 0 (5, 7, 9 dpi) or 1 mM Pi (9 dpi) and harvested at 5, 7, or 9 dpi. Each bar represents three samples (with one exception - only two samples for the 0 Pi, GFP, 9dpi bar). Error bars denote standard deviations. AU - Arbitrary units. B Stability of the BSMV-IPS1 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8, 9: 0 mM Pi, 9 dpi; 10, 11, 12: 1 mM Pi, 9 dpi; 13: plasmid containing BSMVγ-IPS1; 14: water control. M4: GeneRuler 50 bp DNA Ladder (Fermentas); white arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. C Stability of the BSMV-GFP 250 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8: 0 mM Pi, 9 dpi; 9, 10, 11: 1 mM Pi, 9 dpi. D HvIPS1 RNA expression levels in root and shoot tissue determined by qRT-PCR (normalization to ubiquitin). BSMV-IPS1 (black bars), BSMV-GFP 250 (white bars). Plant were grown in 0 mM Pi and harvested at 9 dpi. Each bar represents five samples. Error bars denote standard deviations.
Figure Legend Snippet: Silencing of HvIPS1 in barley roots and shoots . A HvIPS1 RNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants inoculated with BSMV-IPS1 (black bars) or BSMV-GFP 250 (white bars). Plants were grown in 0 (5, 7, 9 dpi) or 1 mM Pi (9 dpi) and harvested at 5, 7, or 9 dpi. Each bar represents three samples (with one exception - only two samples for the 0 Pi, GFP, 9dpi bar). Error bars denote standard deviations. AU - Arbitrary units. B Stability of the BSMV-IPS1 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8, 9: 0 mM Pi, 9 dpi; 10, 11, 12: 1 mM Pi, 9 dpi; 13: plasmid containing BSMVγ-IPS1; 14: water control. M4: GeneRuler 50 bp DNA Ladder (Fermentas); white arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. C Stability of the BSMV-GFP 250 construct in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. Lanes 1, 2, 3: 0 mM Pi, 5 dpi; 4, 5, 6: 0 mM Pi, 7 dpi; 7, 8: 0 mM Pi, 9 dpi; 9, 10, 11: 1 mM Pi, 9 dpi. D HvIPS1 RNA expression levels in root and shoot tissue determined by qRT-PCR (normalization to ubiquitin). BSMV-IPS1 (black bars), BSMV-GFP 250 (white bars). Plant were grown in 0 mM Pi and harvested at 9 dpi. Each bar represents five samples. Error bars denote standard deviations.

Techniques Used: RNA Expression, Quantitative RT-PCR, Construct, Polymerase Chain Reaction, Plasmid Preparation

BSMVγ constructs selected for relative stability also produce successful gene silencing in barley roots . A RT-PCR analysis of the stability of BSMVγ constructs in barley roots. Expected lengths for the PCR products are presented in brackets. Lanes 1, 2: BSMV-IPS1 (493 bp); 3, 4: BSMV-Pht1;1 (610 bp); 5, 6: BSMV-Pht1;4 (616 bp); 7, 8: BSMV-Pht1;7 (623 bp); 9, 10: BSMV-PHR1 (495 bp); 11, 12: BSMV-PHO2 247 (511 bp); 13, 14: BSMV-PHO2 387 (651 bp); 15, 16: GFP 250 (492 bp); 17: control plasmid carrying BSMVγ-GFP 375 (617 bp); 18: control plasmid carrying BSMVγ-IPS1 (493 bp); M4: GeneRuler 50 bp DNA Ladder (Fermentas); M5: O'GeneRuler 50bp DNA Ladder (Fermentas). White arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. B and C HvPHR1 expression levels in root tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHR1 (black bars), BSMV-GFP 250 (white bars). Samples in B are identical to those shown in A, lanes 9, 10 and 15,16. In C, each bar represents five independent samples. AU - arbitrary units; error bars denote standard deviations. D HvPHO2 expression levels in root and leaf tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHO2 247 (black bars), BSMV-GFP 250 (white bars). E As D, but bars represent Pi content in inoculated plants (in μmol Pi/g of fresh weight).
Figure Legend Snippet: BSMVγ constructs selected for relative stability also produce successful gene silencing in barley roots . A RT-PCR analysis of the stability of BSMVγ constructs in barley roots. Expected lengths for the PCR products are presented in brackets. Lanes 1, 2: BSMV-IPS1 (493 bp); 3, 4: BSMV-Pht1;1 (610 bp); 5, 6: BSMV-Pht1;4 (616 bp); 7, 8: BSMV-Pht1;7 (623 bp); 9, 10: BSMV-PHR1 (495 bp); 11, 12: BSMV-PHO2 247 (511 bp); 13, 14: BSMV-PHO2 387 (651 bp); 15, 16: GFP 250 (492 bp); 17: control plasmid carrying BSMVγ-GFP 375 (617 bp); 18: control plasmid carrying BSMVγ-IPS1 (493 bp); M4: GeneRuler 50 bp DNA Ladder (Fermentas); M5: O'GeneRuler 50bp DNA Ladder (Fermentas). White arrow represents DNA fragment of 500 bp; bands below are 400, 300, and 250 bp. B and C HvPHR1 expression levels in root tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHR1 (black bars), BSMV-GFP 250 (white bars). Samples in B are identical to those shown in A, lanes 9, 10 and 15,16. In C, each bar represents five independent samples. AU - arbitrary units; error bars denote standard deviations. D HvPHO2 expression levels in root and leaf tissue determined by qRT-PCR, normalization to 18 S rRNA. BSMV-PHO2 247 (black bars), BSMV-GFP 250 (white bars). E As D, but bars represent Pi content in inoculated plants (in μmol Pi/g of fresh weight).

Techniques Used: Construct, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Expressing, Quantitative RT-PCR

Short inverted repeats are not inherently more stable or efficient for BSMV induced VIGS in barley roots . A HvCel1 RNA expression levels in root tissue determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-Cel1-1, BSMV-Cel1-3, BSMV-Cel1-IR, or BSMV-GFP 375 . Each bar represents five samples, each consisting of one plant in a pot. Error bars denote standard deviations. AU - Arbitrary units. B and C Stability of the BSMV constructs in roots of infected plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. B lanes 1-5: plants infected with BSMV-Cel1-1 (643 bp); lane 6: plasmid containing BSMV-Cel1-1 (643 bp); lane 7: plasmid containing BSMV without insert (242 bp); lanes 8-12: plants infected with BSMV-Cel1-3 (641 bp). C lanes 1-5: plants infected with BSMV-Cel1-IR (388 bp); lane 6: plasmid containing BSMV-Cel1-IR (388 bp); lane 7: plasmid containing BSMV without insert (242 bp); lane 8: plasmid containing BSMV-GFP 375 (617 bp); lanes 9-12: plants infected with BSMV-GFP 375 (617 bp). M5: O'Gene Ruler 50 bp DNA ladder (Fermentas). Expected lengths for the PCR products are given in brackets.
Figure Legend Snippet: Short inverted repeats are not inherently more stable or efficient for BSMV induced VIGS in barley roots . A HvCel1 RNA expression levels in root tissue determined by qRT-PCR (normalization to 18 S rRNA). Plants were inoculated with either BSMV-Cel1-1, BSMV-Cel1-3, BSMV-Cel1-IR, or BSMV-GFP 375 . Each bar represents five samples, each consisting of one plant in a pot. Error bars denote standard deviations. AU - Arbitrary units. B and C Stability of the BSMV constructs in roots of infected plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert. B lanes 1-5: plants infected with BSMV-Cel1-1 (643 bp); lane 6: plasmid containing BSMV-Cel1-1 (643 bp); lane 7: plasmid containing BSMV without insert (242 bp); lanes 8-12: plants infected with BSMV-Cel1-3 (641 bp). C lanes 1-5: plants infected with BSMV-Cel1-IR (388 bp); lane 6: plasmid containing BSMV-Cel1-IR (388 bp); lane 7: plasmid containing BSMV without insert (242 bp); lane 8: plasmid containing BSMV-GFP 375 (617 bp); lanes 9-12: plants infected with BSMV-GFP 375 (617 bp). M5: O'Gene Ruler 50 bp DNA ladder (Fermentas). Expected lengths for the PCR products are given in brackets.

Techniques Used: RNA Expression, Quantitative RT-PCR, Construct, Infection, Polymerase Chain Reaction, Plasmid Preparation

Attempt at silencing HvPht1;1 expression in barley roots . A HvPht1;1 mRNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants were inoculated with either BSMV-Pht1;1 (black bars) or BSMV-GFP 375 (white bars). Plants were grown in hydroponic culture containing 0 or 1 mM Pi. Each bar represents three samples, error bars denote standard deviations. AU - Arbitrary units. B Schematic view of BSMVγ with introduced insert. Arrows show the position of the primers BSMVgbF and BSMVgbR used for assessing stability of insert. The length of virus sequence 5' and 3' of the insert is shown. PCR products of
Figure Legend Snippet: Attempt at silencing HvPht1;1 expression in barley roots . A HvPht1;1 mRNA expression levels in root tissue determined by qRT-PCR (normalization to ubiquitin). Plants were inoculated with either BSMV-Pht1;1 (black bars) or BSMV-GFP 375 (white bars). Plants were grown in hydroponic culture containing 0 or 1 mM Pi. Each bar represents three samples, error bars denote standard deviations. AU - Arbitrary units. B Schematic view of BSMVγ with introduced insert. Arrows show the position of the primers BSMVgbF and BSMVgbR used for assessing stability of insert. The length of virus sequence 5' and 3' of the insert is shown. PCR products of "USER cloning" BSMV vectors are longer by 22 bp compared with restriction enzymes cloning. Not drawn to scale. C Stability of the BSMV-Pht1;1 and BSMV-GFP 375 constructs in roots of inoculated plants. cDNA prepared for qRT-PCR (panel A) was used for PCR with primers flanking the insert as shown in B. Lanes 1, 2, 3: BSMV-Pht1;1, 0 mM Pi (610 bp); 4, 5, 6: BSMV-Pht1;1, 1 mM Pi (610 bp); 7, 8, 9: BSMV-GFP 375 , 0 mM Pi (617 bp); 10, 11, 12: BSMV-GFP 375 ,1 mM Pi (617 bp); 13: plasmid containing BSMVγ-PDS cassette (643 bp); 14: water control; M2: DNA marker; black arrow represents DNA fragment of 564 bp. The expected lengths for PCR products are given in brackets.

Techniques Used: Expressing, Quantitative RT-PCR, Sequencing, Polymerase Chain Reaction, Clone Assay, Construct, Plasmid Preparation, Marker

37) Product Images from "Biochemical and molecular characterization of 3-Methylcrotonylglycinuria in an Italian asymptomatic girl"

Article Title: Biochemical and molecular characterization of 3-Methylcrotonylglycinuria in an Italian asymptomatic girl

Journal: Genetics and Molecular Biology

doi: 10.1590/1678-4685-GMB-2017-0093

Minigene construct and RT-PCR results obtained in expression studies. (A) The minigene construct (pMGene) used in the study: a DNA fragment of approximately 1 kb was directly amplified from the genomic DNA of the patient and cloned into the pMGene as decribed in the Results section. All clones were sequenced, and a wild-type and a mutated clone were used for expression experiments. (B and C) The splicing pattern, evaluated by RT-PCR and sequence analysis of mRNA extracted from cell lines transfected with WT or mutated minigene constructs. Lane 1: molecular weight marker, lane 2: RT-PCR of RNA obtained using the mutant clone; lane 3: RT-PCR of RNA obtained from the normal clone.
Figure Legend Snippet: Minigene construct and RT-PCR results obtained in expression studies. (A) The minigene construct (pMGene) used in the study: a DNA fragment of approximately 1 kb was directly amplified from the genomic DNA of the patient and cloned into the pMGene as decribed in the Results section. All clones were sequenced, and a wild-type and a mutated clone were used for expression experiments. (B and C) The splicing pattern, evaluated by RT-PCR and sequence analysis of mRNA extracted from cell lines transfected with WT or mutated minigene constructs. Lane 1: molecular weight marker, lane 2: RT-PCR of RNA obtained using the mutant clone; lane 3: RT-PCR of RNA obtained from the normal clone.

Techniques Used: Construct, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Clone Assay, Sequencing, Transfection, Molecular Weight, Marker, Mutagenesis

38) Product Images from "A homozygous missense variant in VWA2, encoding an interactor of the Fraser-complex, in a patient with vesicoureteral reflux"

Article Title: A homozygous missense variant in VWA2, encoding an interactor of the Fraser-complex, in a patient with vesicoureteral reflux

Journal: PLoS ONE

doi: 10.1371/journal.pone.0191224

Reduced secretion and intracellular and extracellular aggregation of Arg446Cys VWA2. (a) Cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the One-STrEP-tag. Arrowheads indicate monomeric VWA2. On the right, equal loading is demonstrated by Ponceau staining of the membranes. Asterisks indicate artefact bands. (b) cDNA from non transfected (nt), non-transfected ER stress induced (nt+DTT), wt VWA2 transfected (wt) and Arg446Cys VWA2 (R446C) transfected 293EBNA cells was submitted to RT-PCR and the PCR products separated by agarose gel electrophoresis. Arrows indicate the bands for XBP-1 and ER stress induced XBP-1s. Equal loading is demonstrated by actin control RT-PCR shown below. (c) Equal amounts (0.2 μg) of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the C-terminal fragment (P3) of human VWA2. Under non-reducing conditions higher aggregates are seen. Arrows indicate the border between separation and stacking gel. (d) Equal amounts of cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells as in (a) and of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) as in (b) were separated by agarose-polyacrylamide composite gels under non-reducing conditions and detected with an antibody against the One-STrEP-tag.
Figure Legend Snippet: Reduced secretion and intracellular and extracellular aggregation of Arg446Cys VWA2. (a) Cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the One-STrEP-tag. Arrowheads indicate monomeric VWA2. On the right, equal loading is demonstrated by Ponceau staining of the membranes. Asterisks indicate artefact bands. (b) cDNA from non transfected (nt), non-transfected ER stress induced (nt+DTT), wt VWA2 transfected (wt) and Arg446Cys VWA2 (R446C) transfected 293EBNA cells was submitted to RT-PCR and the PCR products separated by agarose gel electrophoresis. Arrows indicate the bands for XBP-1 and ER stress induced XBP-1s. Equal loading is demonstrated by actin control RT-PCR shown below. (c) Equal amounts (0.2 μg) of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) were separated by SDS-PAGE under reducing and non-reducing conditions and detected with an antibody against the C-terminal fragment (P3) of human VWA2. Under non-reducing conditions higher aggregates are seen. Arrows indicate the border between separation and stacking gel. (d) Equal amounts of cell culture supernatants and cell lysates from wild type (wt) and Arg446Cys VWA2 (R446C) expressing 293EBNA cells as in (a) and of affinity purified wild type (wt) and Arg446Cys VWA2 (R446C) as in (b) were separated by agarose-polyacrylamide composite gels under non-reducing conditions and detected with an antibody against the One-STrEP-tag.

Techniques Used: Cell Culture, Expressing, SDS Page, Strep-tag, Staining, Transfection, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Affinity Purification

39) Product Images from "Multidrug-Resistant Pseudomonas aeruginosa Strain That Caused an Outbreak in a Neurosurgery Ward and Its aac(6′)-Iae Gene Cassette Encoding a Novel Aminoglycoside Acetyltransferase"

Article Title: Multidrug-Resistant Pseudomonas aeruginosa Strain That Caused an Outbreak in a Neurosurgery Ward and Its aac(6′)-Iae Gene Cassette Encoding a Novel Aminoglycoside Acetyltransferase

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.49.9.3734-3742.2005

PCR of class 1 integrons.
Figure Legend Snippet: PCR of class 1 integrons.

Techniques Used: Polymerase Chain Reaction

40) Product Images from "Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation"

Article Title: Elevated Fmr1 mRNA levels and reduced protein expression in a mouse model with an unmethylated Fragile X full mutation

Journal: Experimental cell research

doi: 10.1016/j.yexcr.2006.10.002

PA gel with PCR products of an expanded CGG repeat litter and the breeding couple. The CGG repeat shows both expansion and contraction upon transmission to the next generation.
Figure Legend Snippet: PA gel with PCR products of an expanded CGG repeat litter and the breeding couple. The CGG repeat shows both expansion and contraction upon transmission to the next generation.

Techniques Used: Polymerase Chain Reaction, Transmission Assay

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Countercurrent Chromatography:

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

Clone Assay:

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

Article Title: Sequence Variations of Full-Length Hepatitis B Virus Genomes in Chinese Patients with HBsAg-Negative Hepatitis B Infection
Article Snippet: .. Single genome-length HBV DNA was either retrieved directly by Spe I digestion (New England Biolabs) or further amplified using the Expand High Fidelity PCR system (Roche, Mannheim, Germany) . .. This amplified HBV DNA was then purified and sequenced using sequencing primers that cover the whole viral genome ( ).

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Article Title: Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis
Article Snippet: .. 1 μg of total RNA in the presence of random decamers according to the conditions specified by the manufacturer. cDNAs were amplified by PCR by using the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis, Ind.) in a 50-μl reaction mixture containing 1× Expand HF buffer, 1.5 mM MgCl2 , 200 μM (each) deoxynucleoside triphosphates, 1 μM forward and reverse primers, and 1.5 U of Taq polymerase. .. Subsequently, 2 μl of cDNA generated from mouse tissues or cultured cells was added to the PCR mixture.

Article Title: A Single Nucleotide Polymorphism in 3?-Untranslated Region Contributes to the Regulation of Toll-like Receptor 4 Translation *
Article Snippet: .. Two types of 219-bp genomic fragments (G/G and C/C) of the 3′-UTR of TLR4 at SNP rs11536889 were amplified from two different human genomic DNAs with the Expand High Fidelity PCR system using the primer sets (5′-TGG GAT CCC TCC CCT GTA CCC TTC-3′ (sense) and 5′-CTG GAT CCG TTT CTG AGG AGG CTG GAT G-3′ (antisense)). ..

Polymerase Chain Reaction:

Article Title: Sequence Variations of Full-Length Hepatitis B Virus Genomes in Chinese Patients with HBsAg-Negative Hepatitis B Infection
Article Snippet: .. Single genome-length HBV DNA was either retrieved directly by Spe I digestion (New England Biolabs) or further amplified using the Expand High Fidelity PCR system (Roche, Mannheim, Germany) . .. This amplified HBV DNA was then purified and sequenced using sequencing primers that cover the whole viral genome ( ).

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Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
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Article Title: Persistent Helicobacter pullorum colonization in C57BL/6NTac mice: a new mouse model for an emerging zoonosis
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Article Title: Differences in Innate Immune Responses (In Vitro) to HeLa Cells Infected with Nondisseminating Serovar E and Disseminating Serovar L2 of Chlamydia trachomatis
Article Snippet: .. 1 μg of total RNA in the presence of random decamers according to the conditions specified by the manufacturer. cDNAs were amplified by PCR by using the Expand High Fidelity PCR System (Roche Molecular Biochemicals, Indianapolis, Ind.) in a 50-μl reaction mixture containing 1× Expand HF buffer, 1.5 mM MgCl2 , 200 μM (each) deoxynucleoside triphosphates, 1 μM forward and reverse primers, and 1.5 U of Taq polymerase. .. Subsequently, 2 μl of cDNA generated from mouse tissues or cultured cells was added to the PCR mixture.

Article Title: A Single Nucleotide Polymorphism in 3?-Untranslated Region Contributes to the Regulation of Toll-like Receptor 4 Translation *
Article Snippet: .. Two types of 219-bp genomic fragments (G/G and C/C) of the 3′-UTR of TLR4 at SNP rs11536889 were amplified from two different human genomic DNAs with the Expand High Fidelity PCR system using the primer sets (5′-TGG GAT CCC TCC CCT GTA CCC TTC-3′ (sense) and 5′-CTG GAT CCG TTT CTG AGG AGG CTG GAT G-3′ (antisense)). ..

Construct:

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Reverse Transcription Polymerase Chain Reaction:

Article Title: A multiple-site-specific heteroduplex tracking assay as a tool for the study of viral population dynamics
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Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: .. Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

Mobility Shift:

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
Article Snippet: .. Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions. .. Genomic DNA used for PCRs was isolated from bacterial strains using the Wizard genomic DNA purification kit (Promega).

Quantitative RT-PCR:

Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi
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CTG Assay:

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

Plasmid Preparation:

Article Title: The involvement of replication in single stranded oligonucleotide-mediated gene repair
Article Snippet: .. Plasmid construction DNA inserts for plasmid constructs were amplified by PCR using the Expand High Fidelity PCR system (Roche) and primers linked to restriction enzyme sites suitable for cloning. ..

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    Roche expand high fidelity pcr system
    Transcript levels of cat in B. burgdorferi B31-A3 as measured by <t>QRT-PCR.</t> All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.
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    Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Transcript levels of cat in B. burgdorferi B31-A3 as measured by QRT-PCR. All values have been normalized to the internal control, flaB . Error bars represent standard deviation A. cat transcripts levels were measured in B. burgdorferi A3 harbouring cat reporter plasmids pMB313 (rpoSP 313 fragment), pMB92S (rposP 92S fragment) and pBCAT (vector control) at a cell density of 2 × 10 8 cells ml −1 . Fold changes are relative to the vector control strain. B. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) at varying cell densities. Fold changes are relative to the 2 × 10 7 spirochetes ml −1 culture. C. cat transcripts levels were measured in B. burgdorferi B31-A3 harbouring cat reporter plasmids pMB313 (hatched bars) and pMB92S (black bars) following an increase in growth temperature from 23°C to 34°C. Fold changes are relative to the inoculums used at t = 0 h.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation, Plasmid Preparation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC as cell density increases RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) as spirochete density increased and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS as cell density increased. Fold changes are expressed relative to the initial inoculum. B. QRT-PCR analysis of ospC as cell density increased. Fold changes are expressed relative to the initial inoculum. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with B31-A3 at corresponding cell densities. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared to the B31-A3 at corresponding cell densities. E. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 as cell density increased. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Cell densities are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Transcript levels of cat in B. burgdorferi A3 ntrA and A3 hk2 as measured by QRT-PCR. cat transcripts levels were measured in B. burgdorferi A3 hk2 and A3 ntrA harbouring plasmids pMB313 (hatched bars) and pMB92S (black bars). Fold changes are relative to strains harbouring pBCAT. All values have been normalized to the internal control, flaB . Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Construction of a B. burgdorferi hk2 mutant A. Schematic representation for inactivation of hk2 in B31-A3. hk2 and rrp2 are represented by black arrows as labelled. A DNA fragment harbouring hk2 was PCR amplified using hk2-BF and hk2-BR primers and insertionally disrupted at a unique SphI site with a kanamycin cassette (grey arrow) as described in the Experimental procedures section. Primers are denoted by short black arrows. B. Agarose gel patterns of PCR products for B31-A3 (lane 2) and A3 hk2 (lane 3) using the hk2-BF and hk2-BR primer pair. Disruption of hk2 by the kanamycin cassette resulted in an increased size PCR product (compare lanes 2 and 3). PCR products for the hk2-BF and kan5′ primer pair (lane 4), and the hk2-BR and kan3′ primer pair (lane 5), confirmed the orientation of the kanamycin cassette with respect to hk2 and rrp2 as diagrammed in panel A. RT-PCR analysis with the rrp2-RTF and rrp2-RTR primer pair confirmed the presence of rrp2 transcript in both B31-A3 (lane 6) and A3 hk2 (lane 7). Lane 1 contains DNA markers with the sizes indicated to the left. C. Immunoblot analysis of B31-A3, A3 ntrA and A3 hk2 grown to high cell density (2 × 10 8 cells ml −1 + 24 h). Whole-cell lysates of B. burgdorferi strains equivalent to ∼10 8 cells were separated on a 12% Tris-glycine gel, immobilized on a nitrocellulose membrane and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts and immunoblot analysis of RpoS and OspC following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (grey bars), A3 ntrA (black bars) and A3 hk2 (white bars) grown at 23°C and following a temperature shift to 34°C, and transcripts were quantified using specific primers and probes with the Taqman system. Values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. B. QRT-PCR analysis of ospC following a temperature shift. Fold changes are expressed relative to spirochetes grown at 23°C. C. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 ntrA relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. D. QRT-PCR analysis of rpoS (hatched bars) and ospC (black bars) transcripts in A3 hk2 relative to B31-A3. Fold changes are expressed compared with the B31-A3 at corresponding time points. E. Growth curves of B31-A3 (grey triangles), A3 ntrA (black diamonds) and A3 hk2 (open circles) following a temperature shift from 23°C to 34°C. F. Immunoblot analysis of RpoS and OspC levels in B. burgdorferi strains B31-A3, A3 ntrA and A3 hk2 following an increase in growth temperature from 23°C to 34°C. Whole-cell lysates of B. burgdorferi strains equivalent to approximately 8 × 10 7 −1 × 10 8 cells were separated on 12% Tris-glycine gels, immobilized on nitrocellulose membranes and probed with antiserum specific for the antigens indicated on the left. FlaB serves as a loading control to demonstrate equivalent protein amounts between samples. Time points are indicated at the top of each lane, and positive controls for the A3 ntrA samples are indicated by a plus sign (+).

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

    Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

    Journal: Molecular Microbiology

    Article Title: Insights into the complex regulation of rpoS in Borrelia burgdorferi

    doi: 10.1111/j.1365-2958.2007.05813.x

    Figure Lengend Snippet: Quantitative RT-PCR analysis of rpoS and ospC transcripts following an increase in growth temperature from 23°C to 34°C. RNA was extracted from B. burgdorferi strains B31-A3 (low-passage, white bars) and B31-A (high-passage, black bars) grown at 23°C, and at various time points following a temperature shift to 34°C. Levels of transcripts were measured with specific primer/probe sets using Taqman, and values have been normalized to the internal control, flaB. Data presented represents averages of three assays performed in quadruplicate. Fold changes are expressed relative to spirochetes grown at 23°C. Error bars represent standard deviation. A. QRT-PCR analysis of rpoS following a temperature shift. B. QRT-PCR analysis of ospC following a temperature shift. C. Growth curves of B31-A3 (white squares) and B31-A (black triangles) following a temperature shift from 23 to 34°C.

    Article Snippet: Polymerase chain reaction, RT-PCR, QRT-PCR and DNA mobility-shift assays Polymerase chain reactions were performed using the Expand High Fidelity PCR System (Roche Applied Science, Indianapolis, IN) as per the manufacturer's instructions.

    Techniques: Quantitative RT-PCR, Standard Deviation

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

    Journal: The Journal of Biological Chemistry

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

    doi: 10.1074/jbc.M111.338426

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

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

    Techniques: Expressing, Isolation, Quantitative RT-PCR

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkl852

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

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

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkl852

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

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

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

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

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

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

    doi:

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

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

    Techniques: Polymerase Chain Reaction, Amplification

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

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

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

    doi:

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

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

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

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

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

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

    doi:

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

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

    Techniques: Labeling, Polymerase Chain Reaction

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

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

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

    doi:

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

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

    Techniques: Labeling, Polymerase Chain Reaction, Mutagenesis