Structured Review

TaKaRa puasp svb
Effect of modified forms of the <t>Svb</t> protein on epidermal trichome formation. <t>UAS-GFP</t> (control) ( A,A’ ), UAS-Svb-ACT ( B,B’ ) and UAS-Svb-3Kmut ( C,C’ ) were expressed in the embryonic epidermis under the control of the ptc-Gal4 driver. Top rows show whole embryo cuticles ( A–C ), the bottom row shows close-ups in the ventral region of the third abdominal segment ( A’–C’ ). Svb-ACT, which lacks the N-terminal repressor domain and thus mimics the processed form of Svb, acts as a constitutive activator of transcription and triggers the production of ectopic trichomes. In contrast, Svb-3Kmut -bearing mutations on the 3 Lysines ubiquitinated by Ubr3 in response to Tal peptides- behaves as a repressor and counteracts endogenous Svb activity, resulting in loss of trichomes.
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Images

1) Product Images from "The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning"

Article Title: The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning

Journal: eLife

doi: 10.7554/eLife.39748

Effect of modified forms of the Svb protein on epidermal trichome formation. UAS-GFP (control) ( A,A’ ), UAS-Svb-ACT ( B,B’ ) and UAS-Svb-3Kmut ( C,C’ ) were expressed in the embryonic epidermis under the control of the ptc-Gal4 driver. Top rows show whole embryo cuticles ( A–C ), the bottom row shows close-ups in the ventral region of the third abdominal segment ( A’–C’ ). Svb-ACT, which lacks the N-terminal repressor domain and thus mimics the processed form of Svb, acts as a constitutive activator of transcription and triggers the production of ectopic trichomes. In contrast, Svb-3Kmut -bearing mutations on the 3 Lysines ubiquitinated by Ubr3 in response to Tal peptides- behaves as a repressor and counteracts endogenous Svb activity, resulting in loss of trichomes.
Figure Legend Snippet: Effect of modified forms of the Svb protein on epidermal trichome formation. UAS-GFP (control) ( A,A’ ), UAS-Svb-ACT ( B,B’ ) and UAS-Svb-3Kmut ( C,C’ ) were expressed in the embryonic epidermis under the control of the ptc-Gal4 driver. Top rows show whole embryo cuticles ( A–C ), the bottom row shows close-ups in the ventral region of the third abdominal segment ( A’–C’ ). Svb-ACT, which lacks the N-terminal repressor domain and thus mimics the processed form of Svb, acts as a constitutive activator of transcription and triggers the production of ectopic trichomes. In contrast, Svb-3Kmut -bearing mutations on the 3 Lysines ubiquitinated by Ubr3 in response to Tal peptides- behaves as a repressor and counteracts endogenous Svb activity, resulting in loss of trichomes.

Techniques Used: Modification, Activated Clotting Time Assay, Activity Assay

Schematic representation of Tc-shavenbaby locus ( A ) and transcript ( B ), showing the site at which a GFP-containing marker plasmid was inserted by CRISPR/cas9 genome editing (see also Materials and methods). The mutagenic cassette is inserted into exon 2, that is within the open reading frame upstream of the region encoding the DNA-binding zinc finger domain. Gene disruption leads to mRNA truncation after the insertion site, since Tc-svb expression is absent in homozygous mutants. In addition to segmentation defects and transformation toward thoracic identity, other phenotypes observed in Tc-svb CRISPR mutants include incipient spiracles (possibly a secondary effect of cuticle thinning leading to a defect in the development of tracheal rings); sensory bristles that are shorter and thicker; leg segment boundaries that are not clearly defined; missing leg bristles; unsclerotized pretarsi with soft, rounded apices; and antennae lacking the terminal setae. Therefore, late functions of Tc-svb in epidermal and appendage differentiation are strongly affected in Tc-svb CRISPR mutant embryos, while the segmentation phenotype is milder that Tc-svb -RNAi knockdown due to maternal contribution of Tc-svb (Ray et al., in preparation).
Figure Legend Snippet: Schematic representation of Tc-shavenbaby locus ( A ) and transcript ( B ), showing the site at which a GFP-containing marker plasmid was inserted by CRISPR/cas9 genome editing (see also Materials and methods). The mutagenic cassette is inserted into exon 2, that is within the open reading frame upstream of the region encoding the DNA-binding zinc finger domain. Gene disruption leads to mRNA truncation after the insertion site, since Tc-svb expression is absent in homozygous mutants. In addition to segmentation defects and transformation toward thoracic identity, other phenotypes observed in Tc-svb CRISPR mutants include incipient spiracles (possibly a secondary effect of cuticle thinning leading to a defect in the development of tracheal rings); sensory bristles that are shorter and thicker; leg segment boundaries that are not clearly defined; missing leg bristles; unsclerotized pretarsi with soft, rounded apices; and antennae lacking the terminal setae. Therefore, late functions of Tc-svb in epidermal and appendage differentiation are strongly affected in Tc-svb CRISPR mutant embryos, while the segmentation phenotype is milder that Tc-svb -RNAi knockdown due to maternal contribution of Tc-svb (Ray et al., in preparation).

Techniques Used: Marker, Plasmid Preparation, CRISPR, Binding Assay, Expressing, Transformation Assay, Mutagenesis

2) Product Images from "A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells"

Article Title: A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells

Journal: Scientific Reports

doi: 10.1038/s41598-019-42441-1

Schematic of five putative membrane associated protein phosphatases. Predicted functional domains and protein modification sites are shown for PPM2A (TGGT1_232340), PPM2B (TGGT1_267100), PPM3D (TGGT1_202610), PPM5C (TGGT1_281580), PPM11C (TGGT1_304955). Below PPM11C are the two putative proteins encoded genomic locus of TGGT1_304955. M, myristoylation; P, palmitoylation; SP, signal peptide; TM, transmembrane domain; PP2Cc, PP2C phosphatase catalytic domain.
Figure Legend Snippet: Schematic of five putative membrane associated protein phosphatases. Predicted functional domains and protein modification sites are shown for PPM2A (TGGT1_232340), PPM2B (TGGT1_267100), PPM3D (TGGT1_202610), PPM5C (TGGT1_281580), PPM11C (TGGT1_304955). Below PPM11C are the two putative proteins encoded genomic locus of TGGT1_304955. M, myristoylation; P, palmitoylation; SP, signal peptide; TM, transmembrane domain; PP2Cc, PP2C phosphatase catalytic domain.

Techniques Used: Functional Assay, Modification

3) Product Images from "Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L."

Article Title: Homologous recombination-mediated gene targeting in the liverwort Marchantia polymorpha L.

Journal: Scientific Reports

doi: 10.1038/srep01532

Strategy for targeted disruption of the NOP1 locus and analysis of homologous recombination events. (a) Structure of pJHY-TMp1 and pJHY-CMp1. Only the structure between the left and right borders (LB and RB, respectively) of each vector is shown. DT-A , Diphtheria toxin gene; hpt , hygromycin phosphotransferase gene; Δ En , 3′ part of the maize En element; gus , β-glucuronidase gene. (b) Schematic representation of the genomic structure of the wild-type NOP1 gene region. (c) Structure of pKI406. (d) Structure of the NOP1 locus disrupted by HR. The exons and introns of the NOP1 gene are indicated by blue and open boxes, respectively. The green bar represents the NOP1 flanking region carried by pKI406. Primers are indicated by triangles. Recognition sites of Eco RI and Hin dIII are indicated by vertical lines with the letters E and H, respectively.
Figure Legend Snippet: Strategy for targeted disruption of the NOP1 locus and analysis of homologous recombination events. (a) Structure of pJHY-TMp1 and pJHY-CMp1. Only the structure between the left and right borders (LB and RB, respectively) of each vector is shown. DT-A , Diphtheria toxin gene; hpt , hygromycin phosphotransferase gene; Δ En , 3′ part of the maize En element; gus , β-glucuronidase gene. (b) Schematic representation of the genomic structure of the wild-type NOP1 gene region. (c) Structure of pKI406. (d) Structure of the NOP1 locus disrupted by HR. The exons and introns of the NOP1 gene are indicated by blue and open boxes, respectively. The green bar represents the NOP1 flanking region carried by pKI406. Primers are indicated by triangles. Recognition sites of Eco RI and Hin dIII are indicated by vertical lines with the letters E and H, respectively.

Techniques Used: Homologous Recombination, Plasmid Preparation

4) Product Images from "Functional Roles of FgLaeA in Controlling Secondary Metabolism, Sexual Development, and Virulence in Fusarium graminearum"

Article Title: Functional Roles of FgLaeA in Controlling Secondary Metabolism, Sexual Development, and Virulence in Fusarium graminearum

Journal: PLoS ONE

doi: 10.1371/journal.pone.0068441

Protoperithecia formation and the expression of MAT genes. (A) Protoperithecia formation on carrot agar in the WT (Z3643), Δ FgLaeA , OE ( FgLaeA -overexpression), and add-back strains. Time since perithecia induction (i.e., the removal of aerial mycelia, which had previously grown for 7 days in the dark) is indicated above the pictures. Upper and lower panels for each strain show the surface and undersurface, respectively, of carrot agar plates. The size bar indicates 200 µm. (B) Relative transcript levels for two MAT genes ( x -axis) accumulated in the fungal strains shown in (A) at each of the growth time points (days, y -axis) for total RNA extraction. 3 and 6: days 3 and 6 under vegetative growth, respectively; 8, 9, and 10: days 8, 9, and 10 following perithecial induction, respectively ( i.e. 24 h, 48 h, and 72 h after perithecia induction, respectively). GzRPS16 (FGSG_09438.3) was used as an endogenous control for data normalization [52] . MAT1-1-1 and MAT1-2-1 transcript levels from a 3-day-old vegetative sample of a F. graminearum WT strain were used as references. Statistical analysis was performed with ANOVA and Duncan’s multiple range test. The same letter above bars represents no significant difference.
Figure Legend Snippet: Protoperithecia formation and the expression of MAT genes. (A) Protoperithecia formation on carrot agar in the WT (Z3643), Δ FgLaeA , OE ( FgLaeA -overexpression), and add-back strains. Time since perithecia induction (i.e., the removal of aerial mycelia, which had previously grown for 7 days in the dark) is indicated above the pictures. Upper and lower panels for each strain show the surface and undersurface, respectively, of carrot agar plates. The size bar indicates 200 µm. (B) Relative transcript levels for two MAT genes ( x -axis) accumulated in the fungal strains shown in (A) at each of the growth time points (days, y -axis) for total RNA extraction. 3 and 6: days 3 and 6 under vegetative growth, respectively; 8, 9, and 10: days 8, 9, and 10 following perithecial induction, respectively ( i.e. 24 h, 48 h, and 72 h after perithecia induction, respectively). GzRPS16 (FGSG_09438.3) was used as an endogenous control for data normalization [52] . MAT1-1-1 and MAT1-2-1 transcript levels from a 3-day-old vegetative sample of a F. graminearum WT strain were used as references. Statistical analysis was performed with ANOVA and Duncan’s multiple range test. The same letter above bars represents no significant difference.

Techniques Used: Expressing, Over Expression, RNA Extraction

5) Product Images from "The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity"

Article Title: The Non-Catalytic Domains of Drosophila Katanin Regulate Its Abundance and Microtubule-Disassembly Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0123912

Kat60 lacking the MIT domain does not disassemble microtubules at low levels of accumulation in cells. (A-D) Immunofluorescence microscopy images of Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 0–1.0 (A), 0–1.0 (B), 0–0.01 (C), or 0–0.01 mM CuSO 4 (D) for 20 hours and immunostained for alpha-tubulin and Kat60. Alpha-tubulin and Kat60 images in each panel are displayed with the same scaling. (E) Line graphs of normalized levels of alpha-tubulin as a function of fold overexpression levels of Kat60 for the cells described in A-D. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells with fold overexpression levels of Kat60 below 0. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data represent mean values ± standard deviation from cells with fold overexpression levels of Kat60 between 0 and 40, pooled from six independent experiments (see S3 Table for summary statistics of the single-cell measurements collected).
Figure Legend Snippet: Kat60 lacking the MIT domain does not disassemble microtubules at low levels of accumulation in cells. (A-D) Immunofluorescence microscopy images of Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 0–1.0 (A), 0–1.0 (B), 0–0.01 (C), or 0–0.01 mM CuSO 4 (D) for 20 hours and immunostained for alpha-tubulin and Kat60. Alpha-tubulin and Kat60 images in each panel are displayed with the same scaling. (E) Line graphs of normalized levels of alpha-tubulin as a function of fold overexpression levels of Kat60 for the cells described in A-D. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells with fold overexpression levels of Kat60 below 0. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data represent mean values ± standard deviation from cells with fold overexpression levels of Kat60 between 0 and 40, pooled from six independent experiments (see S3 Table for summary statistics of the single-cell measurements collected).

Techniques Used: Immunofluorescence, Microscopy, Stable Transfection, Expressing, Over Expression, Standard Deviation

Inducible expression of Kat60 results in measurable microtubule disassembly in our single-cell assay. (A and B) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A) or FLAG-Kat80 (B) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A and B were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO 4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S1 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A and B. Molecular weights (in Kd) are shown on the left.
Figure Legend Snippet: Inducible expression of Kat60 results in measurable microtubule disassembly in our single-cell assay. (A and B) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A) or FLAG-Kat80 (B) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A and B were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO 4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S1 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A and B. Molecular weights (in Kd) are shown on the left.

Techniques Used: Expressing, Over Expression, Stable Transfection, Western Blot

Kat60 lacking the MIT domain disassembles microtubules at high levels of accumulation in cells. (A-D) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO 4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S2 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A-D. Molecular weights (in Kd) are shown on the left.
Figure Legend Snippet: Kat60 lacking the MIT domain disassembles microtubules at high levels of accumulation in cells. (A-D) Histograms of normalized levels of alpha-tubulin (Left) and fold overexpression levels of Kat60 (Middle) in Drosophila S2 cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and FLAG-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 0 (light gray), 0.01 (medium gray), 0.1 (dark gray), or 1.0 mM CuSO 4 (black) for 20 hours and immunostained for alpha-tubulin and Kat60. Normalized levels of alpha-tubulin are expressed as a percentage of the mean levels of alpha-tubulin in cells stably expressing GFP alone that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. Fold overexpression levels of Kat60 are expressed as a fraction of the difference in the mean levels of Kat60 between cells stably expressing GFP alone that were treated with control and both Kat60 and Kat80 UTR dsRNA for 7 days total. Data are pooled from three independent experiments (see S2 Table for summary statistics of the single-cell measurements collected). (Right) Immunoblots of cell lysates prepared from the cells described in A-D. Molecular weights (in Kd) are shown on the left.

Techniques Used: Over Expression, Stable Transfection, Expressing, Western Blot

Depletion of Kat80 reduces steady-state Kat60 levels in cells. (A and B) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP alone (A) or GFP and copper-inducible FLAG-Kat80 (B) that were treated with control (lane 1), Kat60 CDS (lane 2), or Kat80 CDS dsRNA (lane 3) for 7 days total. The cells described in B were also treated with 0.1 mM CuSO 4 for 20 hours. Molecular weights (in Kd) are shown on the left.
Figure Legend Snippet: Depletion of Kat80 reduces steady-state Kat60 levels in cells. (A and B) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP alone (A) or GFP and copper-inducible FLAG-Kat80 (B) that were treated with control (lane 1), Kat60 CDS (lane 2), or Kat80 CDS dsRNA (lane 3) for 7 days total. The cells described in B were also treated with 0.1 mM CuSO 4 for 20 hours. Molecular weights (in Kd) are shown on the left.

Techniques Used: Western Blot, Stable Transfection, Expressing

Kat60 lacking the MIT domain or the MIT domain and linker region is not detectably degraded in cells. (A-D) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and Myc-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 1.0 (A), 1.0 (B), 0.1 (C), or 0.01 mM CuSO 4 (D) for 16 hours, washed with S2M, and treated with DMSO (lanes 1–4) or 50 μM MG132 (lanes 5–8) for 0 (lanes 1 and 5), 4 (lanes 2 and 6), 8 (lanes 3 and 7), or 12 hours (lanes 4–8). Molecular weights (in Kd) are shown on the left.
Figure Legend Snippet: Kat60 lacking the MIT domain or the MIT domain and linker region is not detectably degraded in cells. (A-D) Immunoblots of Drosophila S2 cell lysates prepared from cells stably expressing GFP and copper-inducible Kat60 (A), Kat60 and Myc-Kat80 (B), Kat60-ΔMIT (C), or Kat60-AAA (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 1.0 (A), 1.0 (B), 0.1 (C), or 0.01 mM CuSO 4 (D) for 16 hours, washed with S2M, and treated with DMSO (lanes 1–4) or 50 μM MG132 (lanes 5–8) for 0 (lanes 1 and 5), 4 (lanes 2 and 6), 8 (lanes 3 and 7), or 12 hours (lanes 4–8). Molecular weights (in Kd) are shown on the left.

Techniques Used: Western Blot, Stable Transfection, Expressing

Kat60 lacking the MIT domain or the MIT domain and linker region detectably colocalizes with only a few, if any, microtubules in cells. (A-D) TIRF microscopy images of living Drosophila S2 cells stably expressing RFP-alpha-tubulin and copper-inducible GFP-Kat60-K339A (A), GFP-Kat60-K339A and Myc-Kat80 (B), GFP-Kat60-ΔMIT-K339A (C), or GFP-Kat60-AAA-K339A (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 0.1 (A), 0.1 (B), 0.1 (C), or 0.01 mM CuSO 4 (D) for 20 hours. GFP images are displayed with the same scaling.
Figure Legend Snippet: Kat60 lacking the MIT domain or the MIT domain and linker region detectably colocalizes with only a few, if any, microtubules in cells. (A-D) TIRF microscopy images of living Drosophila S2 cells stably expressing RFP-alpha-tubulin and copper-inducible GFP-Kat60-K339A (A), GFP-Kat60-K339A and Myc-Kat80 (B), GFP-Kat60-ΔMIT-K339A (C), or GFP-Kat60-AAA-K339A (D) that were treated with both Kat60 and Kat80 UTR dsRNA for 7 days total. The cells described in A-D were also treated with 0.1 (A), 0.1 (B), 0.1 (C), or 0.01 mM CuSO 4 (D) for 20 hours. GFP images are displayed with the same scaling.

Techniques Used: Microscopy, Stable Transfection, Expressing

6) Product Images from "An ANNEXIN-Like Protein from the Cereal Cyst Nematode Heterodera avenae Suppresses Plant Defense"

Article Title: An ANNEXIN-Like Protein from the Cereal Cyst Nematode Heterodera avenae Suppresses Plant Defense

Journal: PLoS ONE

doi: 10.1371/journal.pone.0122256

Subcellular localization of Ha-ANNEXIN in the plant cell. (A) pUC35S:ANNEXIN:GFP fusion construct and pUC35S:GFP control construct were transformed into onion epidermal cells by bombardment. Scale bar = 100 μm. (B) Agrobacterium tumefaciens cells carrying pCamv35S:ANNEXIN:GFP fusion and pCamv35S:GFP were transiently expressed in Nicotiana benthamiana cells. Scale bar = 20 μm. Western blotting of N . benthamiana leaves infiltrated with pCamv35SGFP-annexin showed expected size of annexin-GFP fusion, which is larger than GFP control. In both (A) and (B), GFP signals were observed in the whole transformed cells for annexin-GFP fusion, which is the same as GFP control.
Figure Legend Snippet: Subcellular localization of Ha-ANNEXIN in the plant cell. (A) pUC35S:ANNEXIN:GFP fusion construct and pUC35S:GFP control construct were transformed into onion epidermal cells by bombardment. Scale bar = 100 μm. (B) Agrobacterium tumefaciens cells carrying pCamv35S:ANNEXIN:GFP fusion and pCamv35S:GFP were transiently expressed in Nicotiana benthamiana cells. Scale bar = 20 μm. Western blotting of N . benthamiana leaves infiltrated with pCamv35SGFP-annexin showed expected size of annexin-GFP fusion, which is larger than GFP control. In both (A) and (B), GFP signals were observed in the whole transformed cells for annexin-GFP fusion, which is the same as GFP control.

Techniques Used: Construct, Transformation Assay, Western Blot

7) Product Images from "Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease"

Article Title: Candida albicans Possesses Sap7 as a Pepstatin A-Insensitive Secreted Aspartic Protease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0032513

Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P
Figure Legend Snippet: Biochemical characteristics of Sap7. (A) SDS-PAGE (left) and western blot (right) analysis of Sap7 with or without EndoH treatment. Analyses of all bands by MALDI-TOF/MS and N-terminal sequencing showed that Sap7 consisted of 2 fragments: fragment 1 (52 kDa) and fragment 2 (15 kDa). Fragment 2 was highly, heterogeneously N -glycosylated, as revealed by EndoH treatment and western blot analysis, which detected the FLAG-tag epitope conjugated at the C-terminal end of Sap7. M: marker, control: protein extracted from the culture supernatant of P. pastoris transformed with a control pHIL-S1 vector. (B) Non-reducing SDS-PAGE analysis. Electrophoretic pattern of non-reduced SDS-PAGE was the same as that of reduced, indicating that the 2 fragments interacted in a non-covalent manner. (C) Primary structure of Sap7. Sap7 was separated into 2 fragments: Fragment 1 was a 52-kDa subunit composed of A144-G440; fragment 2 was a 15-kDa subunit composed of A441-E588. (D) Sensitivity of proteolytic activity to major protease inhibitors. Proteolytic activity was measured using the FRETS-25Ala library with or without various protease inhibitors. While the activity of Sap4 was completely inhibited by pepstatin A, Sap7 did not show sensitivity to any protease inhibitors used here. Averages of at least 3 independent experiments are plotted, and the error bars show S.E.M. ** P

Techniques Used: SDS Page, Western Blot, Mass Spectrometry, Sequencing, FLAG-tag, Marker, Transformation Assay, Plasmid Preparation, Activity Assay

8) Product Images from "Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ"

Article Title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ

Journal: Scientific Reports

doi: 10.1038/srep12728

Interaction of DYRK1A with CDC37-nanoKAZ. ( a ) Structure of the CDC37-nanoKAZ protein. CDC37 and nanoKAZ is fused in-frame with a glycine-serine linker. ( b ) Expression of CDC37-nanoKAZ protein in transfected 293T cells. CDC37-nanoKAZ was detected with antibodies against CDC37 or nanoKAZ. GAPDH was detected as an internal control. ( c ) Interactions of 3xFLAG-DYRK1A and 3xFLAG-DYRK4 with CDC37-nanoKAZ. Luminescence intensities of CDC37-nanoKAZ associated with 3xFLAG-DYRK1A and 3xFLAG-DYRK4 proteins, which were bound on 96-well plates coated with antibody against FLAG, are shown as fold-changes relative to that with EGFP (luminescence due to non-specific binding of CDC37-nanoKAZ). Bar graphs show means ± SD, ***p
Figure Legend Snippet: Interaction of DYRK1A with CDC37-nanoKAZ. ( a ) Structure of the CDC37-nanoKAZ protein. CDC37 and nanoKAZ is fused in-frame with a glycine-serine linker. ( b ) Expression of CDC37-nanoKAZ protein in transfected 293T cells. CDC37-nanoKAZ was detected with antibodies against CDC37 or nanoKAZ. GAPDH was detected as an internal control. ( c ) Interactions of 3xFLAG-DYRK1A and 3xFLAG-DYRK4 with CDC37-nanoKAZ. Luminescence intensities of CDC37-nanoKAZ associated with 3xFLAG-DYRK1A and 3xFLAG-DYRK4 proteins, which were bound on 96-well plates coated with antibody against FLAG, are shown as fold-changes relative to that with EGFP (luminescence due to non-specific binding of CDC37-nanoKAZ). Bar graphs show means ± SD, ***p

Techniques Used: Expressing, Transfection, Binding Assay

Identification of a potent inhibitor of DYRK1A. ( a ) A total of 253 compounds from our original synthetic chemical library were examined. These compounds were used at 4 μM. Relative luminescence intensities are shown. Harmine was used as a positive control and is indicated by the black point. The red point indicates CaNDY. ( b ) Structure of CaNDY. ( c ) CaNDY antagonized the interaction between DYRK1A and CDC37 complex. 293T cells expressing CDC37-nanoKAZ were transiently transfected with an expression vector for 3xFLAG-DYRK1A (Y319F/Y321F) and then treated with the indicated concentrations of CaNDY. The points represent means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) CaNDY inhibited the catalytic activity of DYRK1A in an in vitro kinase assay. Recombinant DYRK1A was incubated with the substrate peptide DYRKtide-F in the presence of the indicated concentrations of small molecules. CaNDY, INDY, and staurosporine inhibited the kinase activity with IC 50 values of 7.9 nM, 122 nM, and 5.4 nM, respectively. Representative dose-response curves with Hill slopes are shown. ( e ) Double-reciprocal plots showing the competitive inhibition of ATP by CaNDY. DYRK1A kinase activity was measured at the indicated concentrations of CaNDY and ATP. Reciprocal velocity was plotted versus 1/[ATP]. K m = 14.1 μM and K i = 1.92 nM.
Figure Legend Snippet: Identification of a potent inhibitor of DYRK1A. ( a ) A total of 253 compounds from our original synthetic chemical library were examined. These compounds were used at 4 μM. Relative luminescence intensities are shown. Harmine was used as a positive control and is indicated by the black point. The red point indicates CaNDY. ( b ) Structure of CaNDY. ( c ) CaNDY antagonized the interaction between DYRK1A and CDC37 complex. 293T cells expressing CDC37-nanoKAZ were transiently transfected with an expression vector for 3xFLAG-DYRK1A (Y319F/Y321F) and then treated with the indicated concentrations of CaNDY. The points represent means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown. ( d ) CaNDY inhibited the catalytic activity of DYRK1A in an in vitro kinase assay. Recombinant DYRK1A was incubated with the substrate peptide DYRKtide-F in the presence of the indicated concentrations of small molecules. CaNDY, INDY, and staurosporine inhibited the kinase activity with IC 50 values of 7.9 nM, 122 nM, and 5.4 nM, respectively. Representative dose-response curves with Hill slopes are shown. ( e ) Double-reciprocal plots showing the competitive inhibition of ATP by CaNDY. DYRK1A kinase activity was measured at the indicated concentrations of CaNDY and ATP. Reciprocal velocity was plotted versus 1/[ATP]. K m = 14.1 μM and K i = 1.92 nM.

Techniques Used: Positive Control, Expressing, Transfection, Plasmid Preparation, Activity Assay, In Vitro, Kinase Assay, Recombinant, Incubation, Inhibition

DYRK1A inhibitors and a HSP90 inhibitor antagonize the interaction of CDC37-nanoKAZ with a DYRK1A mutant. ( a-d ) 293T cells stably expressing CDC37-nanoKAZ were transiently transfected with an expression vector for 3xFLAG-DYRK1A (Y319F/Y321F) and then treated with the indicated concentrations of harmine ( a ), INDY ( b ), staurosporine ( c ), and ganetespib ( d ). Luminescence intensities are shown as fold-changes relative to that at 0 nM, normalized to the amount of 3xFLAG-DYRK1A (Y319F/Y321F) bound on a 96-well plate. Points are means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown.
Figure Legend Snippet: DYRK1A inhibitors and a HSP90 inhibitor antagonize the interaction of CDC37-nanoKAZ with a DYRK1A mutant. ( a-d ) 293T cells stably expressing CDC37-nanoKAZ were transiently transfected with an expression vector for 3xFLAG-DYRK1A (Y319F/Y321F) and then treated with the indicated concentrations of harmine ( a ), INDY ( b ), staurosporine ( c ), and ganetespib ( d ). Luminescence intensities are shown as fold-changes relative to that at 0 nM, normalized to the amount of 3xFLAG-DYRK1A (Y319F/Y321F) bound on a 96-well plate. Points are means ± SD (n = 3). Representative dose-response curves with Hill slopes are shown.

Techniques Used: Mutagenesis, Stable Transfection, Expressing, Transfection, Plasmid Preparation

Structural requirement of CaNDY. ( a ) Structures of PD0439, RD0440, and PD0442. ( b ) In vitro kinase assay using these small molecules, as performed in Fig. 5d . ( c ) CDC37-nanoKAZ binding assay using these small molecules, as performed in Fig. 5c .
Figure Legend Snippet: Structural requirement of CaNDY. ( a ) Structures of PD0439, RD0440, and PD0442. ( b ) In vitro kinase assay using these small molecules, as performed in Fig. 5d . ( c ) CDC37-nanoKAZ binding assay using these small molecules, as performed in Fig. 5c .

Techniques Used: In Vitro, Kinase Assay, Binding Assay

9) Product Images from "Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection"

Article Title: Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection

Journal: BMC Research Notes

doi: 10.1186/s13104-015-1241-6

Schematic representation of the novel negative selection strategy. a Schematic representation of a conditionally cytotoxic gene cassette composed of an SA site, an IRES sequence, a DT - A gene and a polyA sequence. This cassette, when placed upstream of the 5′ arm of an exon-trapping targeting vector, is expected to function as a negative selection marker. b Schematic representation of the impact of the negative selection cassette on integration events. When an exon-trap vector integrates non-homologously into a gene-coding region, the cells are capable of acquiring drug resistance ( i , left panel ). In contrast, when an exon-trap vector possesses the negative selection cassette (ExTraPANS vector), the upstream SA site would trap the splicing from the upstream exon ( grey box ) to allow DT - A gene expression, thereby killing random integrants ( i , right panel ). On the other hand, the presence of the negative selection cassette is expected not to affect homologous recombination-mediated targeted integration ( ii ). Abbreviations are as in Figure 1 .
Figure Legend Snippet: Schematic representation of the novel negative selection strategy. a Schematic representation of a conditionally cytotoxic gene cassette composed of an SA site, an IRES sequence, a DT - A gene and a polyA sequence. This cassette, when placed upstream of the 5′ arm of an exon-trapping targeting vector, is expected to function as a negative selection marker. b Schematic representation of the impact of the negative selection cassette on integration events. When an exon-trap vector integrates non-homologously into a gene-coding region, the cells are capable of acquiring drug resistance ( i , left panel ). In contrast, when an exon-trap vector possesses the negative selection cassette (ExTraPANS vector), the upstream SA site would trap the splicing from the upstream exon ( grey box ) to allow DT - A gene expression, thereby killing random integrants ( i , right panel ). On the other hand, the presence of the negative selection cassette is expected not to affect homologous recombination-mediated targeted integration ( ii ). Abbreviations are as in Figure 1 .

Techniques Used: Selection, Sequencing, Plasmid Preparation, Marker, Expressing, Homologous Recombination

10) Product Images from "Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection"

Article Title: Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection

Journal: BMC Research Notes

doi: 10.1186/s13104-015-1241-6

A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.
Figure Legend Snippet: A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.

Techniques Used: Construct, Clone Assay, Plasmid Preparation, Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification, Flow Cytometry, Marker

11) Product Images from "Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection"

Article Title: Construction and applications of exon-trapping gene-targeting vectors with a novel strategy for negative selection

Journal: BMC Research Notes

doi: 10.1186/s13104-015-1241-6

A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.
Figure Legend Snippet: A simple and efficient method to rapidly construct exon-trapping targeting vectors. a Schematic representation of entry clones with floxed promoterless markers. For simplicity, the plasmid backbone is not drawn. IRES internal ribosome entry site, 2A a 2A-peptide sequence derived from Thosea asigna virus (TaV), Puro R puromycin-resistance gene, Hyg R hygromycin-resistance gene, Neo R neomycin-resistance gene, βgeo lacZ / Neo R , EGFP enhanced green fluorescent protein gene, pA polyadenylation signal. Half-closed triangles and closed triangles represent lox 71 and lox P sequences, respectively. b Primer design for PCR amplification of homology arms. Each primer has four guanine residues at the 5′ end followed by an att B sequence. The four att B sequences att B4, att B1, att B2 and att B3 differ from one another, enabling efficient site-specific BP and LR recombination. The 5′-arm reverse primer should be set on the exon to be trapped (i.e., exon X in panel c ), in order for the 5′-arm fragment to possess an authentic splice acceptor site at the 3′ side. The I- Sce I site added to the 3′-arm reverse primer facilitates linearization of the resulting targeting vector. GSS gene-specific sequences. See text for details. c Flow diagram of construction of targeting vectors based on the MultiSite Gateway system, which consists of three steps: (1) PCR amplification with att B-containing primers, (2) BP recombination between 5′ or 3′ arm fragment and a donor vector (pDONR P4-P1R or pDONR P2R-P3, respectively), and (3) LR recombination to yield the targeting vector by one-time assembly of four DNA fragments ( see text for details). SA splice acceptor site, drug R drug-resistance gene, Km R kanamycin-resistance gene, Amp R ampicillin-resistance gene. d Schematic representation of pENTR SA-IRES-Puro and pENTR SA-IRES-Hyg. These two entry clones harbor an SA site-linked promoterless marker gene. See “ Methods ” for details.

Techniques Used: Construct, Clone Assay, Plasmid Preparation, Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification, Flow Cytometry, Marker

12) Product Images from "Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis"

Article Title: Defining the roles of the N-terminal region and the helicase activity of RECQ4A in DNA repair and homologous recombination in Arabidopsis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt1004

The role of the N-terminus and helicase activity of RECQ4A in suppression of hyper-recombination. The frequency of HR events was determined using the recombination reporter line IC9 and depicted as the relative recombination frequency normalized to the level of recq4A-4 . Each assay was performed at least three times, and the mean values including standard deviations (error bar) are depicted. The expression of the wild-type construct RECQ4A in recq4A-4 mutant background ( recq4A-4 + RECQ4A , green) fully complements the hyper-recombination phenotype of recq4A-4 (A) . Mutant lines transformed with RECQ4A-HD (blue, B ) and RECQ4A-ΔN (red, C ) exhibit an intermediate recombination frequency, which implies a partial complementation of the elevated recombination frequency. The construct RECQ4A-ΔN-HD (grey, D ) cannot complement the hyper-recombination phenotype of recq4A-4 .
Figure Legend Snippet: The role of the N-terminus and helicase activity of RECQ4A in suppression of hyper-recombination. The frequency of HR events was determined using the recombination reporter line IC9 and depicted as the relative recombination frequency normalized to the level of recq4A-4 . Each assay was performed at least three times, and the mean values including standard deviations (error bar) are depicted. The expression of the wild-type construct RECQ4A in recq4A-4 mutant background ( recq4A-4 + RECQ4A , green) fully complements the hyper-recombination phenotype of recq4A-4 (A) . Mutant lines transformed with RECQ4A-HD (blue, B ) and RECQ4A-ΔN (red, C ) exhibit an intermediate recombination frequency, which implies a partial complementation of the elevated recombination frequency. The construct RECQ4A-ΔN-HD (grey, D ) cannot complement the hyper-recombination phenotype of recq4A-4 .

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

The role of the N-terminus and helicase activity of RECQ4A in response to cisplatin and MMS-induced DNA damage. The fresh weight of 10 seedlings after 13 days of genotoxin treatment [5 µM cisplatin (A–D) , 80 ppm MMS (E–H) ] was determined. The relative fresh weight of each line is given as percentage and was calculated from the relation of fresh weight of each line at a respective genotoxin concentration to the fresh weight of the same line without genotoxin treatment. Each assay was performed at least three times as described, and the mean values including standard deviations (error bar) are depicted. The expression of the wild-type construct RECQ4A in recq4A-4 mutant background ( recq4A-4 + RECQ4A , green) enables a full complementation of the elevated sensitivity of recq4A-4 against cisplatin (A) and MMS (E). The constructs RECQ4A-HD (blue, B, F), RECQ4A-ΔN (red, C, G) and RECQ4A-ΔN-HD (grey, D, H) cannot complement the hypersensitivity of recq4A-4 to cisplatin and MMS.
Figure Legend Snippet: The role of the N-terminus and helicase activity of RECQ4A in response to cisplatin and MMS-induced DNA damage. The fresh weight of 10 seedlings after 13 days of genotoxin treatment [5 µM cisplatin (A–D) , 80 ppm MMS (E–H) ] was determined. The relative fresh weight of each line is given as percentage and was calculated from the relation of fresh weight of each line at a respective genotoxin concentration to the fresh weight of the same line without genotoxin treatment. Each assay was performed at least three times as described, and the mean values including standard deviations (error bar) are depicted. The expression of the wild-type construct RECQ4A in recq4A-4 mutant background ( recq4A-4 + RECQ4A , green) enables a full complementation of the elevated sensitivity of recq4A-4 against cisplatin (A) and MMS (E). The constructs RECQ4A-HD (blue, B, F), RECQ4A-ΔN (red, C, G) and RECQ4A-ΔN-HD (grey, D, H) cannot complement the hypersensitivity of recq4A-4 to cisplatin and MMS.

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

Schematic representation of the recombinant RECQ4A variants. (A) The respective RECQ4A constructs were transformed into plant lines. RECQ4A-HD contains an amino acid substitution (K481M, lysine to methionine at position 481) in the Walker A motif of the helicase domain. In RECQ4A-ΔN, the N-terminal amino acids 2–262 of RECQ4A are deleted. RECQ4A-ΔN-HD is truncated in the N-terminus and contains the amino acid substitution in the Walker A motif. (B) The level of conservation of RECQ4A and RECQ4B is indicated by the percentage of identical amino acids in the respective protein region. The chimeric protein RECQ-(4B)4A contains amino acids 1–446 from RECQ4B at the N-terminus adjacent to amino acids 431–1188 of RECQ4A. RECQ-4A(4B) contains N-terminal amino acids 1–969 of RECQ4A and 986–1150 of RECQ4B, including the HRDC domain. Protein sequences of positions of the exchange between RECQ4A and RECQ4B in the chimeric variants are depicted.
Figure Legend Snippet: Schematic representation of the recombinant RECQ4A variants. (A) The respective RECQ4A constructs were transformed into plant lines. RECQ4A-HD contains an amino acid substitution (K481M, lysine to methionine at position 481) in the Walker A motif of the helicase domain. In RECQ4A-ΔN, the N-terminal amino acids 2–262 of RECQ4A are deleted. RECQ4A-ΔN-HD is truncated in the N-terminus and contains the amino acid substitution in the Walker A motif. (B) The level of conservation of RECQ4A and RECQ4B is indicated by the percentage of identical amino acids in the respective protein region. The chimeric protein RECQ-(4B)4A contains amino acids 1–446 from RECQ4B at the N-terminus adjacent to amino acids 431–1188 of RECQ4A. RECQ-4A(4B) contains N-terminal amino acids 1–969 of RECQ4A and 986–1150 of RECQ4B, including the HRDC domain. Protein sequences of positions of the exchange between RECQ4A and RECQ4B in the chimeric variants are depicted.

Techniques Used: Recombinant, Construct, Transformation Assay

13) Product Images from "Characterization of Oyster Voltage-Dependent Anion Channel 2 (VDAC2) Suggests Its Involvement in Apoptosis and Host Defense"

Article Title: Characterization of Oyster Voltage-Dependent Anion Channel 2 (VDAC2) Suggests Its Involvement in Apoptosis and Host Defense

Journal: PLoS ONE

doi: 10.1371/journal.pone.0146049

Interaction between CgVDAC2 and CgBak. (A) Interaction between CgVDAC2 and CgBak detected by the yeast two-hybrid system. The yeast was cultured on respective plates 3–5 days before observed and taken photos. The blue yeast colonies growing on quadruple drop-out SD/-Ade/-His/-Leu/-Trp medium indicated the direct interaction relation between the two proteins. (B) Interaction between CgVDAC2 and CgBak in HEK293 cells detected by co-immunoprecipitation. Flag-tagged CgBak and Myc-tagged CgVDAC2 were co-expressed in HEK293T cells. Co-IP was performed with M2 anti-FLAG antibody. Western blot was carried out with anti-Myc antibody. Empty vector was used as negative control. (Top) CgVDAC2 co-immunoprecipitates with CgBak. (Middle and bottom) The expression of CgVDAC2-Myc and CgBak-Flag proteins.
Figure Legend Snippet: Interaction between CgVDAC2 and CgBak. (A) Interaction between CgVDAC2 and CgBak detected by the yeast two-hybrid system. The yeast was cultured on respective plates 3–5 days before observed and taken photos. The blue yeast colonies growing on quadruple drop-out SD/-Ade/-His/-Leu/-Trp medium indicated the direct interaction relation between the two proteins. (B) Interaction between CgVDAC2 and CgBak in HEK293 cells detected by co-immunoprecipitation. Flag-tagged CgBak and Myc-tagged CgVDAC2 were co-expressed in HEK293T cells. Co-IP was performed with M2 anti-FLAG antibody. Western blot was carried out with anti-Myc antibody. Empty vector was used as negative control. (Top) CgVDAC2 co-immunoprecipitates with CgBak. (Middle and bottom) The expression of CgVDAC2-Myc and CgBak-Flag proteins.

Techniques Used: Cell Culture, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Plasmid Preparation, Negative Control, Expressing

Effects of overexpression of CgVDAC2 on UV irradiation-induced apoptosis in HEK293T cells. (A) Recombinant expression of CgVDAC2 and CgBak in HEK293T cells. The deduced molecular weights of these two proteins are approximate 30 kDa and 26 kDa, respectively. The asterisk indicated a non-specific band. (B) Caspase3 activities of HEK293T cells that expressed distinct recombinant proteins. The Caspase3 activities were determined 24 h after UV irradiation and based on spectrophotometric detection of the chromophore p -nitroaniline ( p NA) after cleavage from the labeled substrate DEVD- p NA. The values are shown as the mean ± S.D (N = 3). Different small letters indicate differences ( P
Figure Legend Snippet: Effects of overexpression of CgVDAC2 on UV irradiation-induced apoptosis in HEK293T cells. (A) Recombinant expression of CgVDAC2 and CgBak in HEK293T cells. The deduced molecular weights of these two proteins are approximate 30 kDa and 26 kDa, respectively. The asterisk indicated a non-specific band. (B) Caspase3 activities of HEK293T cells that expressed distinct recombinant proteins. The Caspase3 activities were determined 24 h after UV irradiation and based on spectrophotometric detection of the chromophore p -nitroaniline ( p NA) after cleavage from the labeled substrate DEVD- p NA. The values are shown as the mean ± S.D (N = 3). Different small letters indicate differences ( P

Techniques Used: Over Expression, Irradiation, Recombinant, Expressing, Labeling

14) Product Images from "Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea"

Article Title: Involvement of luxS in Biofilm Formation by Capnocytophaga ochracea

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147114

Schematic of inactivation and complementation of luxS . (A) Inactivation of luxS by using an ermFermAM cassette. The sequences that flanked the 5′- and 3′- ends of luxS (Coch_1216) were amplified with primers Capno1 and 2, and primers Capno3 and 4, respectively. The ermFermAM cassette was inserted between the amplified fragments and cloned. The plasmid was linearized and introduced into C . ochracea ATCC 27872 by electroporation. The resultant luxS ∷ ermFermAM strain was named TmAI2. (B) Inactivation of luxS by using tetQ . The tetQ fragment was inserted between the sequences that flanked the 5′- and 3′- ends of luxS by using the PCR-based overlap extension method and self-ligattion of the fragment, and then amplified in E . coli . The resultant plasmid was linearized and introduced into C . ochracea ATCC 27872 by electroporation. The resultant luxS ∷ tetQ strain was named LKT7. (C) Complementation of luxS . An ermF-luxS fragment was constructed by using the PCR-based overlap extension method to express both genes under control of the ermF promoter, and the fragment was cloned into the center of the tetQ gene in pAITQ. The resultant plasmid was linearized and introduced into C . ochracea LKT7 by electroporation. The resultant tet ∷ ermF-luxS strain was named luxS-C3.
Figure Legend Snippet: Schematic of inactivation and complementation of luxS . (A) Inactivation of luxS by using an ermFermAM cassette. The sequences that flanked the 5′- and 3′- ends of luxS (Coch_1216) were amplified with primers Capno1 and 2, and primers Capno3 and 4, respectively. The ermFermAM cassette was inserted between the amplified fragments and cloned. The plasmid was linearized and introduced into C . ochracea ATCC 27872 by electroporation. The resultant luxS ∷ ermFermAM strain was named TmAI2. (B) Inactivation of luxS by using tetQ . The tetQ fragment was inserted between the sequences that flanked the 5′- and 3′- ends of luxS by using the PCR-based overlap extension method and self-ligattion of the fragment, and then amplified in E . coli . The resultant plasmid was linearized and introduced into C . ochracea ATCC 27872 by electroporation. The resultant luxS ∷ tetQ strain was named LKT7. (C) Complementation of luxS . An ermF-luxS fragment was constructed by using the PCR-based overlap extension method to express both genes under control of the ermF promoter, and the fragment was cloned into the center of the tetQ gene in pAITQ. The resultant plasmid was linearized and introduced into C . ochracea LKT7 by electroporation. The resultant tet ∷ ermF-luxS strain was named luxS-C3.

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

15) Product Images from "Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing"

Article Title: Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing

Journal: PLoS ONE

doi: 10.1371/journal.pone.0153338

Dissection of B-3-31 (1). The sub-regions of B-3-31 (bent, unbent, 3–1, 3–2, and 3–3; Fig 6B ) were re-cloned into the Asc I site of the IR/MAR vector (pΔBM-d2EGFP-AscI). The resultant plasmids and the control plasmid pΔBM-d2EGFP were transfected into CHO DG44 cells, and transformants were selected with BS. The results of flow-cytometric analysis of d2EGFP expression 30 and 51 days after transfection with the control pΔBM-d2EGFP are shown in A. In the chart, the cell population at 30 days (gray filled line) overlaps with the cell population at 51 days (unfilled line). Changes in average GFP intensity can be observed in the chart. At 51 days, the results of flow-cytometric analysis of d2EGFP expression in various cell populations are shown in B. In each chart, the test cell population (blue filled line) overlaps with the control cell population (pΔBM-d2EGFP; unfilled line). Average GFP intensities were observed for test cells, which can be compared with those of control cells at 51 days (panel A; 91.3). At days 30 and 51 after the transfection, average GFP intensities were divided by that of the control population, and the results are plotted in C. At day 29, cells were fixed and analyzed by FISH using a plasmid-derived probe. To analyze many cells, we evaluated gene amplification of the plasmid in interphase nuclei. Typical FISH images appear in D. Using these images as a standard, the frequency of each type of amplification was scored by examination of more than 300 nuclei in triplicate; the means +/- standard deviations are plotted in D.
Figure Legend Snippet: Dissection of B-3-31 (1). The sub-regions of B-3-31 (bent, unbent, 3–1, 3–2, and 3–3; Fig 6B ) were re-cloned into the Asc I site of the IR/MAR vector (pΔBM-d2EGFP-AscI). The resultant plasmids and the control plasmid pΔBM-d2EGFP were transfected into CHO DG44 cells, and transformants were selected with BS. The results of flow-cytometric analysis of d2EGFP expression 30 and 51 days after transfection with the control pΔBM-d2EGFP are shown in A. In the chart, the cell population at 30 days (gray filled line) overlaps with the cell population at 51 days (unfilled line). Changes in average GFP intensity can be observed in the chart. At 51 days, the results of flow-cytometric analysis of d2EGFP expression in various cell populations are shown in B. In each chart, the test cell population (blue filled line) overlaps with the control cell population (pΔBM-d2EGFP; unfilled line). Average GFP intensities were observed for test cells, which can be compared with those of control cells at 51 days (panel A; 91.3). At days 30 and 51 after the transfection, average GFP intensities were divided by that of the control population, and the results are plotted in C. At day 29, cells were fixed and analyzed by FISH using a plasmid-derived probe. To analyze many cells, we evaluated gene amplification of the plasmid in interphase nuclei. Typical FISH images appear in D. Using these images as a standard, the frequency of each type of amplification was scored by examination of more than 300 nuclei in triplicate; the means +/- standard deviations are plotted in D.

Techniques Used: Dissection, Clone Assay, Plasmid Preparation, Transfection, Flow Cytometry, Expressing, Fluorescence In Situ Hybridization, Derivative Assay, Amplification

Dissection of B-3-31 (2). The B1, B2, and B3 sub-regions of B-3-31 ( Fig 6B ) were re-cloned into the Asc I site of the IR/MAR vector (pΔBM-d2EGFP-AscI). The resultant plasmids and control plasmids with (pΔBM-d2EGFP) or without (pSFV-V d2EGFP) the IR/MAR were transfected into CHO DG44 cells, and transformants were selected with BS. The results of flow-cytometric analysis of d2EGFP expression at 36 and 49 days after transfection with the control pΔBM-d2EGFP plasmid are shown in A. In the chart, the cell population at 36 days (gray filled line) overlaps with the cell population at 49 days (unfilled line). At 49 days, the results of flow-cytometric analysis of d2EGFP expression in various cell populations are shown in B. In each chart, the test cell population (blue filled line) overlaps with the control cell population (pΔBM-d2EGFP; unfilled line). Average GFP intensities are noted for test cells, which can be compared to control cells at 49 days (panel A; 88.7). For two independent cultures from the same transfection, average GFP intensities obtained at the indicated times were divided by that of the control population (C). At day 31, the cells were fixed and analyzed by FISH using a plasmid-derived probe. The frequency of each type of amplification (representative image appears in Fig 7D ) was scored by examining more than 300 nuclei in triplicate; the means +/- standard deviations are plotted in D.
Figure Legend Snippet: Dissection of B-3-31 (2). The B1, B2, and B3 sub-regions of B-3-31 ( Fig 6B ) were re-cloned into the Asc I site of the IR/MAR vector (pΔBM-d2EGFP-AscI). The resultant plasmids and control plasmids with (pΔBM-d2EGFP) or without (pSFV-V d2EGFP) the IR/MAR were transfected into CHO DG44 cells, and transformants were selected with BS. The results of flow-cytometric analysis of d2EGFP expression at 36 and 49 days after transfection with the control pΔBM-d2EGFP plasmid are shown in A. In the chart, the cell population at 36 days (gray filled line) overlaps with the cell population at 49 days (unfilled line). At 49 days, the results of flow-cytometric analysis of d2EGFP expression in various cell populations are shown in B. In each chart, the test cell population (blue filled line) overlaps with the control cell population (pΔBM-d2EGFP; unfilled line). Average GFP intensities are noted for test cells, which can be compared to control cells at 49 days (panel A; 88.7). For two independent cultures from the same transfection, average GFP intensities obtained at the indicated times were divided by that of the control population (C). At day 31, the cells were fixed and analyzed by FISH using a plasmid-derived probe. The frequency of each type of amplification (representative image appears in Fig 7D ) was scored by examining more than 300 nuclei in triplicate; the means +/- standard deviations are plotted in D.

Techniques Used: Dissection, Clone Assay, Plasmid Preparation, Transfection, Flow Cytometry, Expressing, Fluorescence In Situ Hybridization, Derivative Assay, Amplification

16) Product Images from "Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae"

Article Title: Enhancement of protein production via the strong DIT1 terminator and two RNA-binding proteins in Saccharomyces cerevisiae

Journal: Scientific Reports

doi: 10.1038/srep36997

( A ) Growth retardation by overexpression of NAB6 and PAP1 . (Left) Empty pGP564 vector, pGP564-NAB6, pGP564-PAP1, and pGP564-PAP1-NAB6 were separately transformed into the wild-type W303-1A strain (upper; marked NAB6 ) or the nab6 Δ strain (lower; marked nab6 Δ). Two days after inoculation on SD-leucine medium at 30 °C, the colonies were observed. (Right) Optical density (OD) was measured every 30 min from 0 to 24 h after reinoculation at OD 660 = 0.1. The averages of three replicate experiments from 6 to 24 h are indicated. The nab6 Δ/DIT1t strain (gold) and the NAB6 /DIT1t strains harboring pGP564 (white), pGP564-NAB6 (gray), pGP564-NAB6 (blue), or pGP564-PAP1-NAB6 (green) are indicated. ( B ) Identification of the strongest terminator derived from DIT1t . (a) Activation of strong DIT1t -derived terminators by overexpression of NAB6 and PAP1 . The fluorescence intensity of the wild-type DIT1t strain harboring pGP564 was used as the standard. These experiments were conducted independently of those in Fig. 1 . (b) Comparison of terminator activities among DIT1t strains and the standard PGK1t strain in various growth phases. The fluorescence intensities of terminator strains with the GFP gene under the control of the TDH3 promoter were measured by flow cytometry. Sampling times were 6, 12, 24, and 30 h after reinoculation at OD 660 = 0.1. (c) Characterization of the strongest DIT1t -d22 terminator. (Left) Effects of promoter and reporter gene exchange on 3′-UTR activity. These strains expressed either the GFP gene under the control of ACT1pro (left) or the mKO2 gene under the control of TDH3pro (right), as indicated. (Right) Effect of the host strain on 3′-UTR activity. Two wild-type Saccharomyces cerevisiae strains (A451 and TDO2) were transformed individually. The fluorescence intensities of the terminator strains containing the GFP gene under the control of the TDH3 promoter were measured by flow cytometry. The terminator strains examined were PGK1t (purple), DIT1t (orange). DIT1t-m22 (yellow), DIT1t-d7 (gray), DIT1t-d21 (dark gray), and DIT1t -d22 (pink). Data are means ± 1 SD of at least three independent experiments. n.s., not significant; *p
Figure Legend Snippet: ( A ) Growth retardation by overexpression of NAB6 and PAP1 . (Left) Empty pGP564 vector, pGP564-NAB6, pGP564-PAP1, and pGP564-PAP1-NAB6 were separately transformed into the wild-type W303-1A strain (upper; marked NAB6 ) or the nab6 Δ strain (lower; marked nab6 Δ). Two days after inoculation on SD-leucine medium at 30 °C, the colonies were observed. (Right) Optical density (OD) was measured every 30 min from 0 to 24 h after reinoculation at OD 660 = 0.1. The averages of three replicate experiments from 6 to 24 h are indicated. The nab6 Δ/DIT1t strain (gold) and the NAB6 /DIT1t strains harboring pGP564 (white), pGP564-NAB6 (gray), pGP564-NAB6 (blue), or pGP564-PAP1-NAB6 (green) are indicated. ( B ) Identification of the strongest terminator derived from DIT1t . (a) Activation of strong DIT1t -derived terminators by overexpression of NAB6 and PAP1 . The fluorescence intensity of the wild-type DIT1t strain harboring pGP564 was used as the standard. These experiments were conducted independently of those in Fig. 1 . (b) Comparison of terminator activities among DIT1t strains and the standard PGK1t strain in various growth phases. The fluorescence intensities of terminator strains with the GFP gene under the control of the TDH3 promoter were measured by flow cytometry. Sampling times were 6, 12, 24, and 30 h after reinoculation at OD 660 = 0.1. (c) Characterization of the strongest DIT1t -d22 terminator. (Left) Effects of promoter and reporter gene exchange on 3′-UTR activity. These strains expressed either the GFP gene under the control of ACT1pro (left) or the mKO2 gene under the control of TDH3pro (right), as indicated. (Right) Effect of the host strain on 3′-UTR activity. Two wild-type Saccharomyces cerevisiae strains (A451 and TDO2) were transformed individually. The fluorescence intensities of the terminator strains containing the GFP gene under the control of the TDH3 promoter were measured by flow cytometry. The terminator strains examined were PGK1t (purple), DIT1t (orange). DIT1t-m22 (yellow), DIT1t-d7 (gray), DIT1t-d21 (dark gray), and DIT1t -d22 (pink). Data are means ± 1 SD of at least three independent experiments. n.s., not significant; *p

Techniques Used: Over Expression, Plasmid Preparation, Transformation Assay, Derivative Assay, Activation Assay, Fluorescence, Flow Cytometry, Cytometry, Sampling, Activity Assay

Identification of DIT1t -activating factors. ( A ) Sequence of the DIT1 3′-UTR. The length is 208 bp. The cis -element GUUCG is indicated in red. Five deleted regions are indicated by lines. One point mutation (m22) is indicated by an asterisk. Two effective deletion mutations (d7 and d21) are indicated in cyan. ( B ) Genetic interaction between PAP1 and NAB6 . The GFP fluorescence intensity of the control NAB6 strain harboring pGP564 was used as the standard. ( C ) GFP mRNA levels analyzed by RT-PCR. Each total RNA was extracted from the corresponding cells, as denoted in the caption to Fig. 1B. ( D ) Deletion analysis of the DIT1 terminator to identify Pap1p- and Nab6p-recruiting cis region(s). A series of DIT1t mutants (d7 to d21) with 3-bp deletions in the DIT1t region ( Supplementary Table 3 ) and a 10-bp-deleted d2 mutant were constructed. The GFP fluorescence intensity of the wild-type DIT1t strain harboring pGP564 was used as the standard control (indicated as “WT”). A d2-deleted region is indicated by the line. Four deleted regions (d7, d15, d16, and d21) are indicated. ( E ) Gain-of-function analyses using mutated PGK1 terminators. Two strains harboring a mutated terminator ( Supplementary Fig. 7 ) were constructed. The GFP fluorescence intensities were measured. ( F ) Identification of DIT1t -activating cis -element sequences by using point mutagenesis. A series of DIT1t mutants (m1 to m30) with point mutations in the DIT1t region ( Supplementary Table 3 ) were constructed. One point mutation (m22) is indicated by a cyan circle. The GFP fluorescence intensity of the wild-type DIT1t strain harboring pGP564 was used as the standard. Data are means of three or four independent experiments. ( G ) The protein production system involving the cis element GUUCG in the DIT1 3′-UTR. Both Nab6p and Pap1p are considered to be trans -acting factors in this system. The empty control vector (white), pGP564 with NAB6 insert (gray), pGP564 with PAP1 insert (blue), and pGP564 with PAP1 – NAB6 combined insert (green) were separately transformed into the corresponding strains. Data are means ± 1 SD of at least three independent experiments. n.s., not significant; *p
Figure Legend Snippet: Identification of DIT1t -activating factors. ( A ) Sequence of the DIT1 3′-UTR. The length is 208 bp. The cis -element GUUCG is indicated in red. Five deleted regions are indicated by lines. One point mutation (m22) is indicated by an asterisk. Two effective deletion mutations (d7 and d21) are indicated in cyan. ( B ) Genetic interaction between PAP1 and NAB6 . The GFP fluorescence intensity of the control NAB6 strain harboring pGP564 was used as the standard. ( C ) GFP mRNA levels analyzed by RT-PCR. Each total RNA was extracted from the corresponding cells, as denoted in the caption to Fig. 1B. ( D ) Deletion analysis of the DIT1 terminator to identify Pap1p- and Nab6p-recruiting cis region(s). A series of DIT1t mutants (d7 to d21) with 3-bp deletions in the DIT1t region ( Supplementary Table 3 ) and a 10-bp-deleted d2 mutant were constructed. The GFP fluorescence intensity of the wild-type DIT1t strain harboring pGP564 was used as the standard control (indicated as “WT”). A d2-deleted region is indicated by the line. Four deleted regions (d7, d15, d16, and d21) are indicated. ( E ) Gain-of-function analyses using mutated PGK1 terminators. Two strains harboring a mutated terminator ( Supplementary Fig. 7 ) were constructed. The GFP fluorescence intensities were measured. ( F ) Identification of DIT1t -activating cis -element sequences by using point mutagenesis. A series of DIT1t mutants (m1 to m30) with point mutations in the DIT1t region ( Supplementary Table 3 ) were constructed. One point mutation (m22) is indicated by a cyan circle. The GFP fluorescence intensity of the wild-type DIT1t strain harboring pGP564 was used as the standard. Data are means of three or four independent experiments. ( G ) The protein production system involving the cis element GUUCG in the DIT1 3′-UTR. Both Nab6p and Pap1p are considered to be trans -acting factors in this system. The empty control vector (white), pGP564 with NAB6 insert (gray), pGP564 with PAP1 insert (blue), and pGP564 with PAP1 – NAB6 combined insert (green) were separately transformed into the corresponding strains. Data are means ± 1 SD of at least three independent experiments. n.s., not significant; *p

Techniques Used: Sequencing, Mutagenesis, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Construct, Plasmid Preparation, Transformation Assay

17) Product Images from "The H1047R point mutation in p110 alpha changes the morphology of human colon HCT116 cancer cells"

Article Title: The H1047R point mutation in p110 alpha changes the morphology of human colon HCT116 cancer cells

Journal: Cell Death Discovery

doi: 10.1038/cddiscovery.2015.44

The H1047R mutation in the p110 α kinase domain of PI3K affected Bcl-2 expression level. ( a ) Endogenous level of Bcl-2 in HCT116 WT and MUT cells. Endogenous level of Bcl-2 in HCT116 WT and MUT cells was measured by immunoblotting analysis (top). The graph shows the quantification of Bcl-2 bands normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for three individual experiments (bottom). The Bcl-2 level in HCT116 WT cells was at least three times higher than in HCT116 MUT cells. ( b ) Effects of overexpressing WT-p110 α and H1047R-p110 α on Bcl-2 levels. HCT116 WT cells, which were transfected with the Tet-On 3G-inducible plasmids pTRE3G-BI-mCherry/p110 α WT or pTRE3G-BI-mCherry/p110 α H1047R , were cultured in the medium containing indicated concentration of doxycycline (DOX; left). Their Bcl-2 levels were measured by immunoblotting analysis (left). The graph shows the quantifications of Bcl-2 bands of two different clones from the individual cell lines (right). Overexpression of WT-p110 α resulted in a DOX dose-dependent increase in Bcl-2 levels, however, Bcl-2 levels were not verified by overexpression of H1047R-p110 α . ( c ) Effect of p110 α inhibition on Bcl-2 levels. HCT116 WT and MUT cells were serum starved overnight, subsequently cultured in the presence of A66 at the indicated concentrations for 3 h, and then followed by 100 ng/ml EGF stimulation for 20 min. Immunoblotting analysis was used to measure Bcl-2 levels in HCT116 WT (left) and MUT (right) cells. The graph shows the quantifications of expression Bcl-2 levels in HCT116 WT and MUT cells on treatment of A66 (bottom panel). Data are the average of two independent experiments. Bcl-2 levels in HCT116 MUT cells was A66 dose-dependently increased, however, an A66 dose-dependent decrease was observed in HCT WT cells.
Figure Legend Snippet: The H1047R mutation in the p110 α kinase domain of PI3K affected Bcl-2 expression level. ( a ) Endogenous level of Bcl-2 in HCT116 WT and MUT cells. Endogenous level of Bcl-2 in HCT116 WT and MUT cells was measured by immunoblotting analysis (top). The graph shows the quantification of Bcl-2 bands normalized by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for three individual experiments (bottom). The Bcl-2 level in HCT116 WT cells was at least three times higher than in HCT116 MUT cells. ( b ) Effects of overexpressing WT-p110 α and H1047R-p110 α on Bcl-2 levels. HCT116 WT cells, which were transfected with the Tet-On 3G-inducible plasmids pTRE3G-BI-mCherry/p110 α WT or pTRE3G-BI-mCherry/p110 α H1047R , were cultured in the medium containing indicated concentration of doxycycline (DOX; left). Their Bcl-2 levels were measured by immunoblotting analysis (left). The graph shows the quantifications of Bcl-2 bands of two different clones from the individual cell lines (right). Overexpression of WT-p110 α resulted in a DOX dose-dependent increase in Bcl-2 levels, however, Bcl-2 levels were not verified by overexpression of H1047R-p110 α . ( c ) Effect of p110 α inhibition on Bcl-2 levels. HCT116 WT and MUT cells were serum starved overnight, subsequently cultured in the presence of A66 at the indicated concentrations for 3 h, and then followed by 100 ng/ml EGF stimulation for 20 min. Immunoblotting analysis was used to measure Bcl-2 levels in HCT116 WT (left) and MUT (right) cells. The graph shows the quantifications of expression Bcl-2 levels in HCT116 WT and MUT cells on treatment of A66 (bottom panel). Data are the average of two independent experiments. Bcl-2 levels in HCT116 MUT cells was A66 dose-dependently increased, however, an A66 dose-dependent decrease was observed in HCT WT cells.

Techniques Used: Mutagenesis, Expressing, Transfection, Cell Culture, Concentration Assay, Clone Assay, Over Expression, Inhibition

18) Product Images from "Codon usage is an important determinant of gene expression levels largely through its effects on transcription"

Article Title: Codon usage is an important determinant of gene expression levels largely through its effects on transcription

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

doi: 10.1073/pnas.1606724113

Codon optimization results in increased levels of both protein and mRNA in Neurospora . ( A ) Western blot assays showing the effect of codon optimization on protein expression levels in the indicated Neurospora strains. An anti-luciferase antibody is used to detect for the protein levels of LUC, and anti-Myc antibody was used for other Myc-fusion proteins. ( A, Bottom ) A representative Western blot showing protein levels of wt or opt strains. ( A, Top ) Densitometric analyses of three independent samples. The luc , I-SceI , and spa16 genes are driven by the ccg-1 promoter, whereas pkac-1 is under the control of the qa-2 promoter. Membrane was stained and served as loading control. ( B ) Quantitative RT-PCR results showing the relative indicated mRNA levels of wt or optimized opt luc , I-SceI , pkac-1 , and spa16 strain s . ( C ) The relative protein ( Left ) and mRNA ( Right ) levels of wt or optimized opt luc under the control of the frq promoter at the his-3 locus. ( D ) The mRNA levels of wt or opt luc under the control of the frq promoter or the ccg-1 promoter at the his-3 locus. ( E ) The relative protein ( Left ) and mRNA ( Right ) levels of wt or opt spa16 under the control of the vvd promoter at the his-3 locus. The tissues were first cultured in constant darkness for 24 h, then transferred to light for 1 h, and the tissues were harvested. ( F ) The relative protein ( Left ) and mRNA levels of the opt or suboptimized (subopt) luc gene. ( G ) The relative protein ( Left ) and mRNA ( Right ) levels of the opt or subopt I-SceI gene. Error bars shown in all graphs are SDs of the means ( n = 3). ** P
Figure Legend Snippet: Codon optimization results in increased levels of both protein and mRNA in Neurospora . ( A ) Western blot assays showing the effect of codon optimization on protein expression levels in the indicated Neurospora strains. An anti-luciferase antibody is used to detect for the protein levels of LUC, and anti-Myc antibody was used for other Myc-fusion proteins. ( A, Bottom ) A representative Western blot showing protein levels of wt or opt strains. ( A, Top ) Densitometric analyses of three independent samples. The luc , I-SceI , and spa16 genes are driven by the ccg-1 promoter, whereas pkac-1 is under the control of the qa-2 promoter. Membrane was stained and served as loading control. ( B ) Quantitative RT-PCR results showing the relative indicated mRNA levels of wt or optimized opt luc , I-SceI , pkac-1 , and spa16 strain s . ( C ) The relative protein ( Left ) and mRNA ( Right ) levels of wt or optimized opt luc under the control of the frq promoter at the his-3 locus. ( D ) The mRNA levels of wt or opt luc under the control of the frq promoter or the ccg-1 promoter at the his-3 locus. ( E ) The relative protein ( Left ) and mRNA ( Right ) levels of wt or opt spa16 under the control of the vvd promoter at the his-3 locus. The tissues were first cultured in constant darkness for 24 h, then transferred to light for 1 h, and the tissues were harvested. ( F ) The relative protein ( Left ) and mRNA levels of the opt or suboptimized (subopt) luc gene. ( G ) The relative protein ( Left ) and mRNA ( Right ) levels of the opt or subopt I-SceI gene. Error bars shown in all graphs are SDs of the means ( n = 3). ** P

Techniques Used: Western Blot, Expressing, Luciferase, Staining, Quantitative RT-PCR, Cell Culture

19) Product Images from "Tolerance to Excess-Boron Conditions Acquired by Stabilization of a BOR1 Variant with Weak Polarity in Arabidopsis"

Article Title: Tolerance to Excess-Boron Conditions Acquired by Stabilization of a BOR1 Variant with Weak Polarity in Arabidopsis

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2016.00004

Root and shoot growths of transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT grown under a range of boric acid concentrations . The primary root lengths of plants grown for 9 days (A–F) and fresh weights of the aerial portion of plants grown for 20 days (G–L) were determined. Means ± SD are shown ( n = 9–12). Asterisks indicate a significant difference compared with wild-type Col-0 under the same conditions by Student's t -test ( * p
Figure Legend Snippet: Root and shoot growths of transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT grown under a range of boric acid concentrations . The primary root lengths of plants grown for 9 days (A–F) and fresh weights of the aerial portion of plants grown for 20 days (G–L) were determined. Means ± SD are shown ( n = 9–12). Asterisks indicate a significant difference compared with wild-type Col-0 under the same conditions by Student's t -test ( * p

Techniques Used: Transgenic Assay, Expressing

Polar localization of BOR1-GFP-HPT . Transgenic plants expressing BOR1-GFP-HPT were grown on solid medium containing 0.3 μM boric acid for 3 days. (A) BOR1-GFP-HPT in epidermal cells of the meristem zone. GFP (left), FM4-64 (middle), and a merged image (right) are shown. In the merged images, the GFP (green) and FM4-64 (red) overlapping fluorescence signals appear in yellow. (B) BOR1-GFP-HPT in endodermal cells of the differential zone. GFP (left), propidium iodide (middle), and a merged image (right) are shown. Ep, epidermis; Co, cortex; En, endodermis; St, stele. Scale bars represent 25 μm.
Figure Legend Snippet: Polar localization of BOR1-GFP-HPT . Transgenic plants expressing BOR1-GFP-HPT were grown on solid medium containing 0.3 μM boric acid for 3 days. (A) BOR1-GFP-HPT in epidermal cells of the meristem zone. GFP (left), FM4-64 (middle), and a merged image (right) are shown. In the merged images, the GFP (green) and FM4-64 (red) overlapping fluorescence signals appear in yellow. (B) BOR1-GFP-HPT in endodermal cells of the differential zone. GFP (left), propidium iodide (middle), and a merged image (right) are shown. Ep, epidermis; Co, cortex; En, endodermis; St, stele. Scale bars represent 25 μm.

Techniques Used: Transgenic Assay, Expressing, Fluorescence

B-dependent vacuolar sorting of BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT. (A–C) Transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were grown on solid medium containing 0.3 μM boric acid for 4 days and then transferred to solid medium containing 100 μM boric acid. BOR1-GFP-HPT (A) and BOR1(K590A)-GFP-HPT (B) in the root tips grown in 0.3 μM boric acid for 4 days (upper panels), and then shifting to 100 μM boric acid for 3 h (down panels). (C) Quantification of fluorescence intensities of BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT in the plasma membrane after shifting to 100 μM boric acid for 3 h. Means ± SD are shown ( n = 111–149 cells from three roots). (D) Co-localization of BOR1-GFP-HPT and FM4-64 after shifting to 100 μM boric acid for 1.5 h. (E,F) Transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were grown on solid medium containing 3000 μM boric acid for 5 days. Root tip (E) and leaf epidermal cells (F) with BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT. Scale bars represent 25 μm.
Figure Legend Snippet: B-dependent vacuolar sorting of BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT. (A–C) Transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were grown on solid medium containing 0.3 μM boric acid for 4 days and then transferred to solid medium containing 100 μM boric acid. BOR1-GFP-HPT (A) and BOR1(K590A)-GFP-HPT (B) in the root tips grown in 0.3 μM boric acid for 4 days (upper panels), and then shifting to 100 μM boric acid for 3 h (down panels). (C) Quantification of fluorescence intensities of BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT in the plasma membrane after shifting to 100 μM boric acid for 3 h. Means ± SD are shown ( n = 111–149 cells from three roots). (D) Co-localization of BOR1-GFP-HPT and FM4-64 after shifting to 100 μM boric acid for 1.5 h. (E,F) Transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were grown on solid medium containing 3000 μM boric acid for 5 days. Root tip (E) and leaf epidermal cells (F) with BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT. Scale bars represent 25 μm.

Techniques Used: Transgenic Assay, Expressing, Fluorescence

Growth of transgenic plants expressing BOR1-GFP-HPT (A) and BOR1(K590A)-GFP-HPT (B) under a range of boric acid concentrations . Wild-type (Col-0) and transgenic lines were grown on solid media containing 0.3, 30, 3000, and 6000 μM boric acid for 9 days. Scale bars represent 20 mm.
Figure Legend Snippet: Growth of transgenic plants expressing BOR1-GFP-HPT (A) and BOR1(K590A)-GFP-HPT (B) under a range of boric acid concentrations . Wild-type (Col-0) and transgenic lines were grown on solid media containing 0.3, 30, 3000, and 6000 μM boric acid for 9 days. Scale bars represent 20 mm.

Techniques Used: Transgenic Assay, Expressing

B concentrations in roots and shoots of transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT . B concentrations in the roots (A) and aerial portion (B) of the plants were determined after growth on solid media containing 0.3, 3, 30, and 3000 μM boric acid for 14 days. Means ± SD are shown ( n = 4). Asterisks indicate a significant difference compared with wild-type Col-0 under the same conditions by Student's t -test ( * p
Figure Legend Snippet: B concentrations in roots and shoots of transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT . B concentrations in the roots (A) and aerial portion (B) of the plants were determined after growth on solid media containing 0.3, 3, 30, and 3000 μM boric acid for 14 days. Means ± SD are shown ( n = 4). Asterisks indicate a significant difference compared with wild-type Col-0 under the same conditions by Student's t -test ( * p

Techniques Used: Transgenic Assay, Expressing

Comparison of expression levels and patterns of BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT . Transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were grown on solid medium containing 0.3 μM boric acid for 5 days. The images were obtained by a laser scanning confocal microscopy at the same settings for the same tissues of independent lines. The intensities of GFP fluorescence in epidermal cells of leaves (Upper panels) , the root hair zone (Middle panels) , and the root tips (Lower panels) are shown as color-coded heat maps. Scale bars represent 100 μm.
Figure Legend Snippet: Comparison of expression levels and patterns of BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT . Transgenic plants expressing BOR1-GFP-HPT and BOR1(K590A)-GFP-HPT were grown on solid medium containing 0.3 μM boric acid for 5 days. The images were obtained by a laser scanning confocal microscopy at the same settings for the same tissues of independent lines. The intensities of GFP fluorescence in epidermal cells of leaves (Upper panels) , the root hair zone (Middle panels) , and the root tips (Lower panels) are shown as color-coded heat maps. Scale bars represent 100 μm.

Techniques Used: Expressing, Transgenic Assay, Confocal Microscopy, Fluorescence

20) Product Images from "The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination"

Article Title: The Pathogenesis-Related Maize Seed (PRms) Gene Plays a Role in Resistance to Aspergillus flavus Infection and Aflatoxin Contamination

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.01758

Fungal growth in the T1 generation maize kernels. (A) Growth of A. flavus -GFP at 7 dpi (as indicated by relative GFP fluorescence; Rajasekaran et al., 2013 ) in empty vector transformed control and ZmPRms– RNAi transgenic maize kernels (First column: light micrographs and second column: GFP fluorescence micrographs of longitudinal sections of kernels. En: endosperm; Sc: scutellum); (B) Quantification of GFP fluorescence in the kernels of empty vector transformed control and ZmPRms– RNAi maize transgenic plants at 7 days post inoculation (dpi) with A. flavus . Relative fluorescence units (RFU) is directly proportional to fungal growth in the kernels. Data are mean ± SE of 5 biological replicates. ∗∗ Denotes significant difference between control and PRms silenced lines ( P ≤ 0.05).
Figure Legend Snippet: Fungal growth in the T1 generation maize kernels. (A) Growth of A. flavus -GFP at 7 dpi (as indicated by relative GFP fluorescence; Rajasekaran et al., 2013 ) in empty vector transformed control and ZmPRms– RNAi transgenic maize kernels (First column: light micrographs and second column: GFP fluorescence micrographs of longitudinal sections of kernels. En: endosperm; Sc: scutellum); (B) Quantification of GFP fluorescence in the kernels of empty vector transformed control and ZmPRms– RNAi maize transgenic plants at 7 days post inoculation (dpi) with A. flavus . Relative fluorescence units (RFU) is directly proportional to fungal growth in the kernels. Data are mean ± SE of 5 biological replicates. ∗∗ Denotes significant difference between control and PRms silenced lines ( P ≤ 0.05).

Techniques Used: Fluorescence, Plasmid Preparation, Transformation Assay, Transgenic Assay

Confirmation of maize transgenic plants and plant phenotype. (A) Genomic DNA isolated from ZmPRms– RNAi lines and control plant were used to amplify a 1156 bp DNA fragment to confirm the presence of the RNAi cassette (C = empty vector transformed control plants; +ve = plasmid vector used as a PCR template; NEB 2-Log DNA ladder user as a DNA marker); (B) Genomic DNA isolated from ZmPRms– RNAi and empty vector transformed control (C) plants were used to amplify a 433 bp diagnostic DNA fragment to confirm the presence of the ‘ Bar ’ plant selection marker gene (Line 1 = 1–5, Line 3 = 3–5, Line 4 = 4–5; NEB 2-Log DNA ladder user as a DNA marker); (C) Relative expression of the native PRms gene in the kernels of empty vector transformed control and PRms -RNAi maize lines at 7 days post inoculation (dpi) [Gene expression was normalized to the maize ribosomal structural gene GRMZM2G024838 ( Shu et al., 2015 ); data are mean ± SE of 3–4 biological replicates]; (D) Plant growth phenotype (uninfected) of ZmPRms– RNAi lines as compared to the wild type (WT) or empty vector transformed control transgenic maize plants.
Figure Legend Snippet: Confirmation of maize transgenic plants and plant phenotype. (A) Genomic DNA isolated from ZmPRms– RNAi lines and control plant were used to amplify a 1156 bp DNA fragment to confirm the presence of the RNAi cassette (C = empty vector transformed control plants; +ve = plasmid vector used as a PCR template; NEB 2-Log DNA ladder user as a DNA marker); (B) Genomic DNA isolated from ZmPRms– RNAi and empty vector transformed control (C) plants were used to amplify a 433 bp diagnostic DNA fragment to confirm the presence of the ‘ Bar ’ plant selection marker gene (Line 1 = 1–5, Line 3 = 3–5, Line 4 = 4–5; NEB 2-Log DNA ladder user as a DNA marker); (C) Relative expression of the native PRms gene in the kernels of empty vector transformed control and PRms -RNAi maize lines at 7 days post inoculation (dpi) [Gene expression was normalized to the maize ribosomal structural gene GRMZM2G024838 ( Shu et al., 2015 ); data are mean ± SE of 3–4 biological replicates]; (D) Plant growth phenotype (uninfected) of ZmPRms– RNAi lines as compared to the wild type (WT) or empty vector transformed control transgenic maize plants.

Techniques Used: Transgenic Assay, Isolation, Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Marker, Diagnostic Assay, Selection, Expressing

Aflatoxin (AF) contents in the control (empty vector transformed) and ZmPRms– RNAi transgenic maize kernels at 7 days post inoculation (dpi) with A. flavus . (A) AFB1; and (B) AFB2. Data are mean ± SE of 4 biological replicates. ∗∗ Denotes significant difference between control and PRms silenced lines ( P ≤ 0.05).
Figure Legend Snippet: Aflatoxin (AF) contents in the control (empty vector transformed) and ZmPRms– RNAi transgenic maize kernels at 7 days post inoculation (dpi) with A. flavus . (A) AFB1; and (B) AFB2. Data are mean ± SE of 4 biological replicates. ∗∗ Denotes significant difference between control and PRms silenced lines ( P ≤ 0.05).

Techniques Used: Plasmid Preparation, Transformation Assay, Transgenic Assay

Relative expression of predicted ZmPRms -regulated downstream target (obtained from gene regulatory network analysis; Figure 5 and Table 1 ) candidate genes in the control (empty vector transformed) and ZmPRms– RNAi transgenic maize kernels at 7 days post inoculation (dpi) with A. flavus . Gene expression was normalized to the maize ribosomal structural gene GRMZM2G024838 ( Shu et al., 2015 ). Data are mean ± SE of 3–4 biological replicates. ∗∗ / ∗ Denote significant difference between control and PRms silenced lines ( ∗∗ P ≤ 0.05, ∗ P ≤ 0.1). Un1 (Uncharacterized 1; GRMZM2G042752), Un2 (Uncharacterized 2; GRMZM2G061398), Un3 (Uncharacterized 3; GRMZM2G092415), Un4 (Uncharacterized 4; GRMZM2G101412), Un6 (Uncharacterized 6; GRMZM2G383338).
Figure Legend Snippet: Relative expression of predicted ZmPRms -regulated downstream target (obtained from gene regulatory network analysis; Figure 5 and Table 1 ) candidate genes in the control (empty vector transformed) and ZmPRms– RNAi transgenic maize kernels at 7 days post inoculation (dpi) with A. flavus . Gene expression was normalized to the maize ribosomal structural gene GRMZM2G024838 ( Shu et al., 2015 ). Data are mean ± SE of 3–4 biological replicates. ∗∗ / ∗ Denote significant difference between control and PRms silenced lines ( ∗∗ P ≤ 0.05, ∗ P ≤ 0.1). Un1 (Uncharacterized 1; GRMZM2G042752), Un2 (Uncharacterized 2; GRMZM2G061398), Un3 (Uncharacterized 3; GRMZM2G092415), Un4 (Uncharacterized 4; GRMZM2G101412), Un6 (Uncharacterized 6; GRMZM2G383338).

Techniques Used: Expressing, Plasmid Preparation, Transformation Assay, Transgenic Assay

ZmPRms gene and vector construction. (A) Genomic structure of the Zea mays ( Zm ) PRms gene; (B) Amino acid sequence of the ZmPRms gene; (C) Vector diagram of the RNAi construct used for maize transformation to silence the ZmPRms gene (Abbreviations: 27 Zn = maize endosperm-specific promoter, RB = right border, LB = left border, PR10 = intron (maize), Ubi 1 = constitutive promoter, Bar = bialaphos resistance gene, ocs and nos = transcription terminators).
Figure Legend Snippet: ZmPRms gene and vector construction. (A) Genomic structure of the Zea mays ( Zm ) PRms gene; (B) Amino acid sequence of the ZmPRms gene; (C) Vector diagram of the RNAi construct used for maize transformation to silence the ZmPRms gene (Abbreviations: 27 Zn = maize endosperm-specific promoter, RB = right border, LB = left border, PR10 = intron (maize), Ubi 1 = constitutive promoter, Bar = bialaphos resistance gene, ocs and nos = transcription terminators).

Techniques Used: Plasmid Preparation, Sequencing, Construct, Transformation Assay

21) Product Images from "The Specialized Roles in Carotenogenesis and Apocarotenogenesis of the Phytoene Synthase Gene Family in Saffron"

Article Title: The Specialized Roles in Carotenogenesis and Apocarotenogenesis of the Phytoene Synthase Gene Family in Saffron

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2019.00249

Three dimensional models and location of the saffron phytoene synthase enzymes. (A) Three-dimensional models of the CsPSY enzymes. The models for CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 were created using the PPM Server ( http://opm.phar.umich.edu/server.php ). The α-helices, and loops are depicted as pink and blue, respectively. Blue dots indicate the membrane surface. (B) Subcellular localization of GFP fusion proteins of CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 in agro-infiltrated tobacco leaves after 5 days as detected with confocal laser scanning microscopy and enhanced green fluorescent protein (eGFP) expression. Chlorophyll auto-fluorescence in red (left panel), eGFP fluorescence is shown in green (middle panel) and a merged overlay of the eGFP/chlorophyll fluorescence (right panel) is shown in yellow.
Figure Legend Snippet: Three dimensional models and location of the saffron phytoene synthase enzymes. (A) Three-dimensional models of the CsPSY enzymes. The models for CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 were created using the PPM Server ( http://opm.phar.umich.edu/server.php ). The α-helices, and loops are depicted as pink and blue, respectively. Blue dots indicate the membrane surface. (B) Subcellular localization of GFP fusion proteins of CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 in agro-infiltrated tobacco leaves after 5 days as detected with confocal laser scanning microscopy and enhanced green fluorescent protein (eGFP) expression. Chlorophyll auto-fluorescence in red (left panel), eGFP fluorescence is shown in green (middle panel) and a merged overlay of the eGFP/chlorophyll fluorescence (right panel) is shown in yellow.

Techniques Used: Confocal Laser Scanning Microscopy, Expressing, Fluorescence

Functional complementation of the saffron PSY proteins. (A) Complex models of the CsPSY enzymes and their co-factor. (B) Escherichia coli cells harboring the pAC-85b vector were additionally transformed with saffron PSY constructs or empty vector. Cells carrying the pAtPSY vector confer accumulation of β-carotene and were used as a positive control. HPLC chromatograms for the extracted pigments are shown. The peak representing β-carotene was observed in cells with CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 constructs, but not in the empty vector. The inset shows the absorption spectrum of the β-carotene peak.
Figure Legend Snippet: Functional complementation of the saffron PSY proteins. (A) Complex models of the CsPSY enzymes and their co-factor. (B) Escherichia coli cells harboring the pAC-85b vector were additionally transformed with saffron PSY constructs or empty vector. Cells carrying the pAtPSY vector confer accumulation of β-carotene and were used as a positive control. HPLC chromatograms for the extracted pigments are shown. The peak representing β-carotene was observed in cells with CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 constructs, but not in the empty vector. The inset shows the absorption spectrum of the β-carotene peak.

Techniques Used: Functional Assay, Plasmid Preparation, Transformation Assay, Construct, Positive Control, High Performance Liquid Chromatography

22) Product Images from "mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases"

Article Title: mRNA Deadenylation Is Coupled to Translation Rates by the Differential Activities of Ccr4-Not Nucleases

Journal: Molecular Cell

doi: 10.1016/j.molcel.2018.05.033

Codon Optimality Influences Pab1 Association and mRNA Deadenylation Rate (A) Plot of Pab1-bound mRNA levels relative to total mRNA levels following normalization to poly(A) tail length and binning of mRNAs according to codon optimality. Values were calculated using previously published Pab1 RNA immunoprecipitation sequencing (RIP-seq), total RNA sequencing (RNA-seq), and poly(A) tail length profiling by sequencing (PAL-seq) data. ∗∗∗ p adj
Figure Legend Snippet: Codon Optimality Influences Pab1 Association and mRNA Deadenylation Rate (A) Plot of Pab1-bound mRNA levels relative to total mRNA levels following normalization to poly(A) tail length and binning of mRNAs according to codon optimality. Values were calculated using previously published Pab1 RNA immunoprecipitation sequencing (RIP-seq), total RNA sequencing (RNA-seq), and poly(A) tail length profiling by sequencing (PAL-seq) data. ∗∗∗ p adj

Techniques Used: Immunoprecipitation, Sequencing, RNA Sequencing Assay

Shortening of Pab1-Bound Poly(A) Tails Is Catalyzed by Ccr4 (A) Deadenylation of a 23-mer-A30 RNA in the absence or presence of Pab1 by Ccr4-Not and variant complexes with mutations in the active site of Ccr4 (Ccr4-inactive), Caf1 (Caf1-inactive), or both Ccr4 and Caf1 (double-inactive). Densitometric analyses were performed on selected gels (bottom). (B) Global poly(A) tail length in wild-type (WT) S. cerevisiae and strains containing deletion of CCR4 or CAF1 . The red asterisk indicates incomplete deadenylation in the ccr4 Δ strain. Densitometric analyses were performed on selected gels (bottom). (C) Deadenylation of a 23-mer-A30 RNA by isolated Caf1 protein, Ccr4 (EEP nuclease domain), or the Caf1-Ccr4 heterodimer. (D) Coomassie-stained SDS-PAGE of pull-down assays showing binding of purified Ccr4 or Caf1 to immobilized GST-Pab1. Contaminant proteins are indicated with asterisks. .
Figure Legend Snippet: Shortening of Pab1-Bound Poly(A) Tails Is Catalyzed by Ccr4 (A) Deadenylation of a 23-mer-A30 RNA in the absence or presence of Pab1 by Ccr4-Not and variant complexes with mutations in the active site of Ccr4 (Ccr4-inactive), Caf1 (Caf1-inactive), or both Ccr4 and Caf1 (double-inactive). Densitometric analyses were performed on selected gels (bottom). (B) Global poly(A) tail length in wild-type (WT) S. cerevisiae and strains containing deletion of CCR4 or CAF1 . The red asterisk indicates incomplete deadenylation in the ccr4 Δ strain. Densitometric analyses were performed on selected gels (bottom). (C) Deadenylation of a 23-mer-A30 RNA by isolated Caf1 protein, Ccr4 (EEP nuclease domain), or the Caf1-Ccr4 heterodimer. (D) Coomassie-stained SDS-PAGE of pull-down assays showing binding of purified Ccr4 or Caf1 to immobilized GST-Pab1. Contaminant proteins are indicated with asterisks. .

Techniques Used: Variant Assay, Isolation, Staining, SDS Page, Binding Assay, Purification

Models for Pab1 Release by Ccr4 and Coupling of Translation and Deadenylation Rates by Caf1 (A) Proposed model for the organization of Pab1 on the poly(A) tail with RRMs depicted linearly. The Pab1 molecule proximal to the 3′ UTR binds ∼22 adenosines through RRMs 1−3, and distal Pab1 molecules bind ∼28 adenosines with RRMs 1–4. Naked poly(A) not bound by Pab1 can be removed by either Caf1 or Ccr4, while RNA within the binding site of Pab1 can only be accessed by Ccr4. Pab1 self-association and interaction with other proteins may lead to higher-order structures on RNA. (B) The modular architecture of Pab1 permits deadenylation to occur before it completely dissociates from the poly(A) tail. (C) Translation elongation rate may contribute to Pab1 occupancy to affect deadenylation rate. Ccr4 is required for deadenylation of all mRNAs, but the requirement for Caf1 is specific to mRNAs with low codon optimality or reduced Pab1 occupancy.
Figure Legend Snippet: Models for Pab1 Release by Ccr4 and Coupling of Translation and Deadenylation Rates by Caf1 (A) Proposed model for the organization of Pab1 on the poly(A) tail with RRMs depicted linearly. The Pab1 molecule proximal to the 3′ UTR binds ∼22 adenosines through RRMs 1−3, and distal Pab1 molecules bind ∼28 adenosines with RRMs 1–4. Naked poly(A) not bound by Pab1 can be removed by either Caf1 or Ccr4, while RNA within the binding site of Pab1 can only be accessed by Ccr4. Pab1 self-association and interaction with other proteins may lead to higher-order structures on RNA. (B) The modular architecture of Pab1 permits deadenylation to occur before it completely dissociates from the poly(A) tail. (C) Translation elongation rate may contribute to Pab1 occupancy to affect deadenylation rate. Ccr4 is required for deadenylation of all mRNAs, but the requirement for Caf1 is specific to mRNAs with low codon optimality or reduced Pab1 occupancy.

Techniques Used: Binding Assay

Pab1 Organization on the Poly(A) Tail (A) Deadenylation by Ccr4-inactive Ccr4-Not to map Pab1-binding site on A30 and 23-mer-A30 RNA substrates. Red asterisks indicate accumulated product poly(A) tail lengths. A and S4B. Models of Pab1 binding to each RNA are shown on the right. (C) Deadenylation by Ccr4-inactive Ccr4-Not on 20-mer-A60 RNA in the absence or presence of Pab1 (2:1 molar ratio to RNA). Densitometric analysis of the reaction with Pab1 shows that the protected RNA fragment is ∼50–55 adenosines. A model for Pab1-RNA binding is shown. .
Figure Legend Snippet: Pab1 Organization on the Poly(A) Tail (A) Deadenylation by Ccr4-inactive Ccr4-Not to map Pab1-binding site on A30 and 23-mer-A30 RNA substrates. Red asterisks indicate accumulated product poly(A) tail lengths. A and S4B. Models of Pab1 binding to each RNA are shown on the right. (C) Deadenylation by Ccr4-inactive Ccr4-Not on 20-mer-A60 RNA in the absence or presence of Pab1 (2:1 molar ratio to RNA). Densitometric analysis of the reaction with Pab1 shows that the protected RNA fragment is ∼50–55 adenosines. A model for Pab1-RNA binding is shown. .

Techniques Used: Binding Assay, RNA Binding Assay

Pab1 Stimulates Stepwise Deadenylation by Ccr4-Not (A) Deadenylation by purified Ccr4-Not in the presence and absence of Pab1. The RNA substrate comprises 20 non-poly(A) nucleotides followed by a 60-adenosine poly(A) tail. RNA products (4-min time points) were resolved on a denaturing polyacrylamide gel. Pab1-bound substrates were prepared with two Pab1 molecules per RNA. (B) Coomassie-stained SDS-PAGE of pull-down assay showing binding of purified Ccr4-Not (red labels) to immobilized GST-Pab1. Purified proteins (before mixing), Input (proteins mixed before loading on resin), and Pull-down (proteins bound to resin after washing) are shown. The asterisk indicates a contaminant protein. (C) Deadenylation of 5′ fluorescently labeled 23-mer-A30 RNA substrate. Pab1-bound substrates were prepared with one Pab1 molecule per RNA. Poly(A) tail lengths are indicated, and RRM footprints are marked with red asterisks. (D) Deadenylation of 5′ fluorescently labeled 23-mer-A30 RNA substrates in the presence of Pab1 variants. The positions of footprints observed with wild-type Pab1 in (C) are indicated with red asterisks. .
Figure Legend Snippet: Pab1 Stimulates Stepwise Deadenylation by Ccr4-Not (A) Deadenylation by purified Ccr4-Not in the presence and absence of Pab1. The RNA substrate comprises 20 non-poly(A) nucleotides followed by a 60-adenosine poly(A) tail. RNA products (4-min time points) were resolved on a denaturing polyacrylamide gel. Pab1-bound substrates were prepared with two Pab1 molecules per RNA. (B) Coomassie-stained SDS-PAGE of pull-down assay showing binding of purified Ccr4-Not (red labels) to immobilized GST-Pab1. Purified proteins (before mixing), Input (proteins mixed before loading on resin), and Pull-down (proteins bound to resin after washing) are shown. The asterisk indicates a contaminant protein. (C) Deadenylation of 5′ fluorescently labeled 23-mer-A30 RNA substrate. Pab1-bound substrates were prepared with one Pab1 molecule per RNA. Poly(A) tail lengths are indicated, and RRM footprints are marked with red asterisks. (D) Deadenylation of 5′ fluorescently labeled 23-mer-A30 RNA substrates in the presence of Pab1 variants. The positions of footprints observed with wild-type Pab1 in (C) are indicated with red asterisks. .

Techniques Used: Purification, Staining, SDS Page, Pull Down Assay, Binding Assay, Labeling

23) Product Images from "Cell Type Diversity in Hepatitis B Virus RNA Splicing and Its Regulation"

Article Title: Cell Type Diversity in Hepatitis B Virus RNA Splicing and Its Regulation

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.00207

Identification of cis -regulatory elements involved in HBV 3.5-kb RNA splicing in cell-type dependent and -independent manners. (A) A schematic representation of constructs and PCR primers used in the intron-swapping experiment is indicated. (B) HBV WT and HBV/int-SV40 were expressed in HepG2 and HEK293 cells. PCR products were analyzed by agarose gel electrophoresis (left). Schematic of unspliced and spliced forms detected is shown (right). (C) SV40-L WT and SV40/int-HBV were expressed in the cells and PCR products were analyzed as shown in (B) .
Figure Legend Snippet: Identification of cis -regulatory elements involved in HBV 3.5-kb RNA splicing in cell-type dependent and -independent manners. (A) A schematic representation of constructs and PCR primers used in the intron-swapping experiment is indicated. (B) HBV WT and HBV/int-SV40 were expressed in HepG2 and HEK293 cells. PCR products were analyzed by agarose gel electrophoresis (left). Schematic of unspliced and spliced forms detected is shown (right). (C) SV40-L WT and SV40/int-HBV were expressed in the cells and PCR products were analyzed as shown in (B) .

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

Characterization of the intronic sequence important for silencing of the 3.5 kb RNA splicing. (A) A schematic representation of mutated HBV genomes derived from GT-C with deletions and/or substitutions within the major intron region is shown. Putative secondary structures predicted by the CentroidFold ( http://rtools.cbrc.jp/centroidfold/ ) in nt 2858–2983 and 3197-48 regions are indicated. Each predicted base pair is colored with the heat color gradation from blue to red corresponding to the base-pairing probability. Bold lines are drawn along the sequences targeting to introduce substitution mutations S1 and S2. (B) RT-PCR analysis of HBV RNAs expressed from WT and a series of deletion-mutated HBV genomes (D1–D5) in HepG2 and HuH-7 cells. The quantity ratio of the spliced RNAs to the unspliced RNA (Sp ratio) was determined. ND, not determined. Open arrows, unspliced forms. ∗ cDNA products with aberrant sizes. (C) As described in (B) but HBV mutated genomes S1/D1 and S1/D1/S2 in addition to WT and D1 were used for RNA expression. (D) As in (C) but S1 mutant and WT were expressed in the cells. Bands corresponding to unspliced 3.5 kb RNA and its spliced forms (Sp RNA) were indicated.
Figure Legend Snippet: Characterization of the intronic sequence important for silencing of the 3.5 kb RNA splicing. (A) A schematic representation of mutated HBV genomes derived from GT-C with deletions and/or substitutions within the major intron region is shown. Putative secondary structures predicted by the CentroidFold ( http://rtools.cbrc.jp/centroidfold/ ) in nt 2858–2983 and 3197-48 regions are indicated. Each predicted base pair is colored with the heat color gradation from blue to red corresponding to the base-pairing probability. Bold lines are drawn along the sequences targeting to introduce substitution mutations S1 and S2. (B) RT-PCR analysis of HBV RNAs expressed from WT and a series of deletion-mutated HBV genomes (D1–D5) in HepG2 and HuH-7 cells. The quantity ratio of the spliced RNAs to the unspliced RNA (Sp ratio) was determined. ND, not determined. Open arrows, unspliced forms. ∗ cDNA products with aberrant sizes. (C) As described in (B) but HBV mutated genomes S1/D1 and S1/D1/S2 in addition to WT and D1 were used for RNA expression. (D) As in (C) but S1 mutant and WT were expressed in the cells. Bands corresponding to unspliced 3.5 kb RNA and its spliced forms (Sp RNA) were indicated.

Techniques Used: Sequencing, Derivative Assay, Introduce, Reverse Transcription Polymerase Chain Reaction, RNA Expression, Mutagenesis

Effect of deletions in intron region of the HBV genome on the splicing efficiency in human HCC and non-hepatic cells. RT-PCR analyses of HBV RNAs expressed from WT and deletion-mutants corresponding to D1 and D4 derived from GT-A (deleted regions; nt 2990–3202 in D1, nt 2864-48 in D4), -C (nt 2984–3196 in D1, nt 2858-48 in D4) and -D (nt 2951–3163 in D1, nt 2858-48 in D4) in human HCC (HepG2, HuH-7 and PLC/PRF/5) cells (A) and in non-hepatic (HEK293, A549, and HeLa) cells (B) . Sp ratio; the quantity ratio of the spliced RNAs to the unspliced RNA. Open arrows, unspliced forms.
Figure Legend Snippet: Effect of deletions in intron region of the HBV genome on the splicing efficiency in human HCC and non-hepatic cells. RT-PCR analyses of HBV RNAs expressed from WT and deletion-mutants corresponding to D1 and D4 derived from GT-A (deleted regions; nt 2990–3202 in D1, nt 2864-48 in D4), -C (nt 2984–3196 in D1, nt 2858-48 in D4) and -D (nt 2951–3163 in D1, nt 2858-48 in D4) in human HCC (HepG2, HuH-7 and PLC/PRF/5) cells (A) and in non-hepatic (HEK293, A549, and HeLa) cells (B) . Sp ratio; the quantity ratio of the spliced RNAs to the unspliced RNA. Open arrows, unspliced forms.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Planar Chromatography

24) Product Images from "High levels of TopBP1 induce ATR-dependent shut-down of rRNA transcription and nucleolar segregation"

Article Title: High levels of TopBP1 induce ATR-dependent shut-down of rRNA transcription and nucleolar segregation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv371

Expression of ectopic TopBP1 induces shut-down of rRNA transcription and nucleolar segregation. ( A ) Expression of eGFP-TopBP1 was left non-induced (eGFP-TopBP1 Off), induced for 24 h (eGFP-TopBP1 On), or cells were treated with ActD. Nascent transcripts were labelled with a short pulse of FUrd. Confocal images of FUrd, endogenous RNA Pol I, UBF, NPM (Nucleophosmin) and NCL (Nucleolin) and eGFP-TopBP1 are shown. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. ( B ) Pre-rRNA synthesis levels were determined with qRT-PCR from eGFP-TopBP1 cells left non-induced (Off), induced for the indicated times or treated with ActD. Percentages of pre-rRNA levels normalized to ACTB transcription are shown relative to non-induced cells.
Figure Legend Snippet: Expression of ectopic TopBP1 induces shut-down of rRNA transcription and nucleolar segregation. ( A ) Expression of eGFP-TopBP1 was left non-induced (eGFP-TopBP1 Off), induced for 24 h (eGFP-TopBP1 On), or cells were treated with ActD. Nascent transcripts were labelled with a short pulse of FUrd. Confocal images of FUrd, endogenous RNA Pol I, UBF, NPM (Nucleophosmin) and NCL (Nucleolin) and eGFP-TopBP1 are shown. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. ( B ) Pre-rRNA synthesis levels were determined with qRT-PCR from eGFP-TopBP1 cells left non-induced (Off), induced for the indicated times or treated with ActD. Percentages of pre-rRNA levels normalized to ACTB transcription are shown relative to non-induced cells.

Techniques Used: Expressing, Quantitative RT-PCR

ATR is required for TopBP1-induced nucleolar segregation. ( A ) Chemical inhibition of ATR and expression of ATR activation mutant of TopBP1 attenuate nucleolar segregation induced by ectopic TopBP1. Expression of eGFP-TopBP1 (wild-type, WT or W1145R mutant) was induced for 15 h alone, or in the presence of ATR inhibitor ETP-46464 (ATRi), or cells expressing eGFP-TopBP1 (WT or W1145R, as indicated) were treated with ActD. Nascent transcripts were labelled with FUrd. Confocal images of FUrd, endogenous RNA Pol I and Nucleolin (NCL) and eGFP-TopBP1 are shown. ( B ) Downregulation of ATR suppresses nucleolar segregation induced by eGFP-TopBP1. Cells were transfected with ATR siRNA, with unspecific negative siRNA (NEG siRNA) or left non-transfected. Thereafter, expression of eGFP-TopBP1 WT was induced for 24 h prior to fixation, or left non-induced. Percentage of cells with eGFP-TopBP1 foci is shown with representative wide-field microscopy images of eGFP-TopBP1. Results are means of three independent experiments. Over 100 nuclei were counted in each experiment. Standard deviations are shown. Scale bars are 10 μm. ( C ) Immunoblot of the whole-cell extract shows levels of eGFP-TopBP1 and ATR. β-Tubulin is shown as control of protein loading.
Figure Legend Snippet: ATR is required for TopBP1-induced nucleolar segregation. ( A ) Chemical inhibition of ATR and expression of ATR activation mutant of TopBP1 attenuate nucleolar segregation induced by ectopic TopBP1. Expression of eGFP-TopBP1 (wild-type, WT or W1145R mutant) was induced for 15 h alone, or in the presence of ATR inhibitor ETP-46464 (ATRi), or cells expressing eGFP-TopBP1 (WT or W1145R, as indicated) were treated with ActD. Nascent transcripts were labelled with FUrd. Confocal images of FUrd, endogenous RNA Pol I and Nucleolin (NCL) and eGFP-TopBP1 are shown. ( B ) Downregulation of ATR suppresses nucleolar segregation induced by eGFP-TopBP1. Cells were transfected with ATR siRNA, with unspecific negative siRNA (NEG siRNA) or left non-transfected. Thereafter, expression of eGFP-TopBP1 WT was induced for 24 h prior to fixation, or left non-induced. Percentage of cells with eGFP-TopBP1 foci is shown with representative wide-field microscopy images of eGFP-TopBP1. Results are means of three independent experiments. Over 100 nuclei were counted in each experiment. Standard deviations are shown. Scale bars are 10 μm. ( C ) Immunoblot of the whole-cell extract shows levels of eGFP-TopBP1 and ATR. β-Tubulin is shown as control of protein loading.

Techniques Used: Inhibition, Expressing, Activation Assay, Mutagenesis, Transfection, Microscopy

eGFP-TopBP1 associates with chromatin, localizes predominantly in nucleoli in permeabilized cells and binds to the transcribed region of the rDNA repeat. ( A ) Cells were left non-induced or induced to express eGFP-TopBP1, WT or W1145R, as indicated. Cells were left unfractionated (WCE, whole cell extract) or fractionated in soluble/cytoplasmic (S), chromatin bound (B) or matrix (M) fractions as described in ‘Materials and Methods’ section and subjected to immunoblotting. UBF is shown as a control for protein binding to chromatin, Lamin A/C as a nuclear matrix protein and β-Tubulin as a cytoplasmic protein. ( B ) Cells induced to express eGFP-TopBP1 WT or W1145R, were treated with detergent before fixing. Wide-field images show endogenous Nucleolin (NCL) or eGFP-TopBP1. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. ( C ) Schematic presentation of a complete rDNA repeat unit (U13369) that shows transcribed region (boxed area), 5′ and 3′ external transcribed spacers (ETS), internal transcribed spacers (ITS1 and ITS2), the source of 18S, 5.8S and 28S rRNAs, and the non-transcribed spacer (NTS). Positions of primer pairs H1, H4, H13, H18, H27 and H32 at the rRNA gene are shown. All positions and lengths of elements are in scale. ( D ) Quantification of ChIP with anti-TopBP1 (left) and anti-RNA Pol I (right) IPs in assays using nuclear material from normal U2OS cells or from cells that were induced to express eGFP-TopBP1. DNA was quantitated by qPCR using primer pairs indicated in (C). Quantification is presented as percentages of input material precipitated. Mean values of three independent experiments are shown with standard deviations.
Figure Legend Snippet: eGFP-TopBP1 associates with chromatin, localizes predominantly in nucleoli in permeabilized cells and binds to the transcribed region of the rDNA repeat. ( A ) Cells were left non-induced or induced to express eGFP-TopBP1, WT or W1145R, as indicated. Cells were left unfractionated (WCE, whole cell extract) or fractionated in soluble/cytoplasmic (S), chromatin bound (B) or matrix (M) fractions as described in ‘Materials and Methods’ section and subjected to immunoblotting. UBF is shown as a control for protein binding to chromatin, Lamin A/C as a nuclear matrix protein and β-Tubulin as a cytoplasmic protein. ( B ) Cells induced to express eGFP-TopBP1 WT or W1145R, were treated with detergent before fixing. Wide-field images show endogenous Nucleolin (NCL) or eGFP-TopBP1. Merge shows an overlay of panels (DNA excluded). Scale bar is 10 μm. ( C ) Schematic presentation of a complete rDNA repeat unit (U13369) that shows transcribed region (boxed area), 5′ and 3′ external transcribed spacers (ETS), internal transcribed spacers (ITS1 and ITS2), the source of 18S, 5.8S and 28S rRNAs, and the non-transcribed spacer (NTS). Positions of primer pairs H1, H4, H13, H18, H27 and H32 at the rRNA gene are shown. All positions and lengths of elements are in scale. ( D ) Quantification of ChIP with anti-TopBP1 (left) and anti-RNA Pol I (right) IPs in assays using nuclear material from normal U2OS cells or from cells that were induced to express eGFP-TopBP1. DNA was quantitated by qPCR using primer pairs indicated in (C). Quantification is presented as percentages of input material precipitated. Mean values of three independent experiments are shown with standard deviations.

Techniques Used: Protein Binding, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

Endogenous TopBP1 co-localizes with RNA pol I. Cells were left non-treated or were treated with ActD. Wide-field images of endogenous TopBP1 and RNA pol I are shown. Percentages (± standard deviation, three independent experiments) show the proportion of cells that showed co-localization of RNA pol I and TopBP1 foci. Over 100 nuclei were counted in each experiment. Scale bar is 10 μm.
Figure Legend Snippet: Endogenous TopBP1 co-localizes with RNA pol I. Cells were left non-treated or were treated with ActD. Wide-field images of endogenous TopBP1 and RNA pol I are shown. Percentages (± standard deviation, three independent experiments) show the proportion of cells that showed co-localization of RNA pol I and TopBP1 foci. Over 100 nuclei were counted in each experiment. Scale bar is 10 μm.

Techniques Used: Standard Deviation

Both endogenous and ectopic TopBP1 display nucleolar localization. eGFP-TopBP1 was left non-induced (Off) or induced (On) for 24 h before fixing the cells for immunoelectron microscopy. TopBP1 was immunostained with 10 nm, and RNA pol I, UBF and NCL with 5 nm gold particles. ( A ) Examples of nucleoli in a non-induced and an induced cell at lower magnification. ( B ) Higher magnification of nucleoli in non-induced and induced cells. Endogenous TopBP1 partly co-localizes with RNA Pol I, UBF and NCL in the electron-dense regions of the nucleolus, but in induced cells it strongly concentrates in distinct regions immediately adjacent to main body of nucleolus (arrowheads). Scale bars are 100, 500 or 1000 nm, as indicated.
Figure Legend Snippet: Both endogenous and ectopic TopBP1 display nucleolar localization. eGFP-TopBP1 was left non-induced (Off) or induced (On) for 24 h before fixing the cells for immunoelectron microscopy. TopBP1 was immunostained with 10 nm, and RNA pol I, UBF and NCL with 5 nm gold particles. ( A ) Examples of nucleoli in a non-induced and an induced cell at lower magnification. ( B ) Higher magnification of nucleoli in non-induced and induced cells. Endogenous TopBP1 partly co-localizes with RNA Pol I, UBF and NCL in the electron-dense regions of the nucleolus, but in induced cells it strongly concentrates in distinct regions immediately adjacent to main body of nucleolus (arrowheads). Scale bars are 100, 500 or 1000 nm, as indicated.

Techniques Used: Immuno-Electron Microscopy

TopBP1-induced nucleolar segregation is not associated with cell cycle arrest. ( A ) Cells were treated with ActD for 2 h and let recover for indicated times, or induced to express eGFP-TopBP1 for the same times. Nascent DNA was labelled with a short pulse of EdU with subsequent immunolabelling of eGFP-TopBP1 before the cells were fixed and microscoped. Scale bar is 50 μm. ( B ) Immunoblots of TopBP1, phosphorylated Chk1, total Chk1, phosphorylated p53, total p53 and p21. β-Tubulin served as a control of protein loading. ( C ) Localization of activated p53 in cells expressing eGFP-TopBP1 and in cells treated with ActD. Cells were left non-induced or induced with doxycycline to express eGFP-TopBP1 for 24 h, or treated for 2 h with 30 nM ActD and fixed. Activated p53 (DO-1) and Rad9 were immunolabelled. Scale bar is 10 μm. ( D ) Fractions of p53-positive cells were determined in non-treated (Ctrl), ActD-treated (as in A, assayed 24 h after release) and in Nutlin 3-a-treated (5 μM, 3 h) cell populations. From induced (DOX) samples, those expressing (e+) eGFP-TopBP1 and not expressing (e-) were scored separately. p53 positive cells were identified automatically based on a threshold established in the nutlin-3a treated cells. Mean values of three independent experiments are shown with standard deviations. Statistical significance ( P -value ≤0.05) was calculated with ANOVA and Dunnett's T3 post hoc tests.
Figure Legend Snippet: TopBP1-induced nucleolar segregation is not associated with cell cycle arrest. ( A ) Cells were treated with ActD for 2 h and let recover for indicated times, or induced to express eGFP-TopBP1 for the same times. Nascent DNA was labelled with a short pulse of EdU with subsequent immunolabelling of eGFP-TopBP1 before the cells were fixed and microscoped. Scale bar is 50 μm. ( B ) Immunoblots of TopBP1, phosphorylated Chk1, total Chk1, phosphorylated p53, total p53 and p21. β-Tubulin served as a control of protein loading. ( C ) Localization of activated p53 in cells expressing eGFP-TopBP1 and in cells treated with ActD. Cells were left non-induced or induced with doxycycline to express eGFP-TopBP1 for 24 h, or treated for 2 h with 30 nM ActD and fixed. Activated p53 (DO-1) and Rad9 were immunolabelled. Scale bar is 10 μm. ( D ) Fractions of p53-positive cells were determined in non-treated (Ctrl), ActD-treated (as in A, assayed 24 h after release) and in Nutlin 3-a-treated (5 μM, 3 h) cell populations. From induced (DOX) samples, those expressing (e+) eGFP-TopBP1 and not expressing (e-) were scored separately. p53 positive cells were identified automatically based on a threshold established in the nutlin-3a treated cells. Mean values of three independent experiments are shown with standard deviations. Statistical significance ( P -value ≤0.05) was calculated with ANOVA and Dunnett's T3 post hoc tests.

Techniques Used: Western Blot, Expressing

Deletion of BRCT domains 0–2 or 4–5 abrogates nucleolar segregation induced by TopBP1. ( A ) Schematic picture of TopBP1 deletion mutant constructs. eGFP, enhanced green fluorescent protein tag; 0–8, BRCT domains 0–8; AAD, ATR activation domain; NLS, nuclear localization signal; FL, full-length. BRCT domains 0–2, 3, 4–5, 6 or 7–8 were deleted in D0–2, D3, D4–5, D6 and D7–8, respectively. All elements are in scale. ( B ) eGFP-TopBP1 constructs were transiently transfected into U2OS cells. Wide-field images show endogenous UBF or eGFP-TopBP1. ( C ) Cells were left non-induced, induced to express eGFP-TopBP1 or left non-induced and treated with ActD. Cells were immunostained for endogenous Rad9, Hus1 or RNA pol I. Non-treated and doxycycline panels are confocal images and ActD is a wide-field image. Merge shows an overlay of panels (DNA excluded). Scale bars are 10 μm.
Figure Legend Snippet: Deletion of BRCT domains 0–2 or 4–5 abrogates nucleolar segregation induced by TopBP1. ( A ) Schematic picture of TopBP1 deletion mutant constructs. eGFP, enhanced green fluorescent protein tag; 0–8, BRCT domains 0–8; AAD, ATR activation domain; NLS, nuclear localization signal; FL, full-length. BRCT domains 0–2, 3, 4–5, 6 or 7–8 were deleted in D0–2, D3, D4–5, D6 and D7–8, respectively. All elements are in scale. ( B ) eGFP-TopBP1 constructs were transiently transfected into U2OS cells. Wide-field images show endogenous UBF or eGFP-TopBP1. ( C ) Cells were left non-induced, induced to express eGFP-TopBP1 or left non-induced and treated with ActD. Cells were immunostained for endogenous Rad9, Hus1 or RNA pol I. Non-treated and doxycycline panels are confocal images and ActD is a wide-field image. Merge shows an overlay of panels (DNA excluded). Scale bars are 10 μm.

Techniques Used: Mutagenesis, Construct, Activation Assay, Transfection

25) Product Images from "Characterization of novel endo-β-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG"

Article Title: Characterization of novel endo-β-N-acetylglucosaminidases from Sphingobacterium species, Beauveria bassiana and Cordyceps militaris that specifically hydrolyze fucose-containing oligosaccharides and human IgG

Journal: Scientific Reports

doi: 10.1038/s41598-017-17467-y

Confirmation of oligosaccharide structure on rituximab after ENGase treatment. LC-MS analysis was performed on the following samples: ( A ) rituximab without ENGase treatment ( B ) treated with ORF1188 and ( C ) treated with Cordyceps ENGase. Note that the single peak derived from GlcNAc-fucose was observed in samples ( B ) and ( C ), but not in sample ( A ).
Figure Legend Snippet: Confirmation of oligosaccharide structure on rituximab after ENGase treatment. LC-MS analysis was performed on the following samples: ( A ) rituximab without ENGase treatment ( B ) treated with ORF1188 and ( C ) treated with Cordyceps ENGase. Note that the single peak derived from GlcNAc-fucose was observed in samples ( B ) and ( C ), but not in sample ( A ).

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Derivative Assay

Sequence alignment of ORF1188, Beauveria and Cordyceps ENGases. Alignment of amino acid sequences of ORF1188, Beauveria and Cordyceps ENGases was shown. Beauveria and Cordyceps ENGases are consisted of 315 amino acid residues and they respectively show 54% and 53% sequence similarities to the ORF1188 ENGase.
Figure Legend Snippet: Sequence alignment of ORF1188, Beauveria and Cordyceps ENGases. Alignment of amino acid sequences of ORF1188, Beauveria and Cordyceps ENGases was shown. Beauveria and Cordyceps ENGases are consisted of 315 amino acid residues and they respectively show 54% and 53% sequence similarities to the ORF1188 ENGase.

Techniques Used: Sequencing

Characterization of Beauveria and Cordyceps ENGases. ( A ) Recombinant Beauveria and Cordyceps ENGases were expressed in E. coli and were purified. Protein samples (0.5 μg of each) were then subjected to SDS-PAGE using a 5–20% acrylamide gel, following which the gel was stained with CBB. ( B ) Effects of pH on the enzymatic activity of Beauveria and Cordyceps ENGases. The assay was carried out using 100 mM buffers at various pHs. ( C ) Rituximab (IgG) was incubated separately with Endo-S, Endo-CC1, recombinant Beauveria ENGase and recombinant Cordyceps ENGase overnight and subsequently analyzed by SDS-PAGE. The CBB stained gel shows the heavy chain of IgG is migrating as a protein with MW of approximately 50 kDa. ( D ) RNase B was incubated separately with Endo-S, Endo-CC1, recombinant Beauveria ENGase and recombinant Cordyceps ENGase overnight and subsequently analyzed by SDS-PAGE.
Figure Legend Snippet: Characterization of Beauveria and Cordyceps ENGases. ( A ) Recombinant Beauveria and Cordyceps ENGases were expressed in E. coli and were purified. Protein samples (0.5 μg of each) were then subjected to SDS-PAGE using a 5–20% acrylamide gel, following which the gel was stained with CBB. ( B ) Effects of pH on the enzymatic activity of Beauveria and Cordyceps ENGases. The assay was carried out using 100 mM buffers at various pHs. ( C ) Rituximab (IgG) was incubated separately with Endo-S, Endo-CC1, recombinant Beauveria ENGase and recombinant Cordyceps ENGase overnight and subsequently analyzed by SDS-PAGE. The CBB stained gel shows the heavy chain of IgG is migrating as a protein with MW of approximately 50 kDa. ( D ) RNase B was incubated separately with Endo-S, Endo-CC1, recombinant Beauveria ENGase and recombinant Cordyceps ENGase overnight and subsequently analyzed by SDS-PAGE.

Techniques Used: Recombinant, Purification, SDS Page, Acrylamide Gel Assay, Staining, Activity Assay, Incubation

26) Product Images from "Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana"

Article Title: Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.02062

Contribution of structural domains to PCD induction by three candidate Heterodera avenae effectors in Nicotiana benthamiana . (A) Schematic of the structural domains of the three genes isotig12969 , isotig19390 and isotig16511 . The former two genes contain PROF (profilin) domains, and the third gene encodes a SP (indicated in red) and an endonuclease domain (NUC, indicated in blue). (B) Visualization of the phenotypes associated with transient expression of the three genes and their respective gene fragments lacking the structural domains. The first number below the gene name indicates the total number of necrosis spots, and the second number below the gene name indicates the total number of infiltration spots. Western blotting confirmed the expression of isotig16511Δ. (C) Necrosis indices of the infiltration spots of the three genes and their respective gene fragments lacking the structural domains. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of the gene fragments lacking the structural domains compared to those of the intact genes ( P
Figure Legend Snippet: Contribution of structural domains to PCD induction by three candidate Heterodera avenae effectors in Nicotiana benthamiana . (A) Schematic of the structural domains of the three genes isotig12969 , isotig19390 and isotig16511 . The former two genes contain PROF (profilin) domains, and the third gene encodes a SP (indicated in red) and an endonuclease domain (NUC, indicated in blue). (B) Visualization of the phenotypes associated with transient expression of the three genes and their respective gene fragments lacking the structural domains. The first number below the gene name indicates the total number of necrosis spots, and the second number below the gene name indicates the total number of infiltration spots. Western blotting confirmed the expression of isotig16511Δ. (C) Necrosis indices of the infiltration spots of the three genes and their respective gene fragments lacking the structural domains. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of the gene fragments lacking the structural domains compared to those of the intact genes ( P

Techniques Used: Expressing, Western Blot, Standard Deviation

Candidate Heterodera avenae effectors (example isotig18549) suppress cell death triggered by other candidate H. avenae effectors (example isotig12969) in Nicotiana benthamiana . (A) Assay of the suppression of isotig12969-triggered cell death in N. benthamiana by isotig18549. The results of the verification of gene expression of isotig18549 and isotig12969 by western blotting are shown below. (B) Necrosis index of isotig18549 and control eGFP followed by isotig12969. Each column shows the mean and standard deviation.
Figure Legend Snippet: Candidate Heterodera avenae effectors (example isotig18549) suppress cell death triggered by other candidate H. avenae effectors (example isotig12969) in Nicotiana benthamiana . (A) Assay of the suppression of isotig12969-triggered cell death in N. benthamiana by isotig18549. The results of the verification of gene expression of isotig18549 and isotig12969 by western blotting are shown below. (B) Necrosis index of isotig18549 and control eGFP followed by isotig12969. Each column shows the mean and standard deviation.

Techniques Used: Expressing, Western Blot, Standard Deviation

Symptoms of systemic transient expression of Heterodera avenae effectors in Nicotiana benthamiana . (A) Untreated wild plant. (B) Empty vector control. (C) eGFP control. (D) Severe necrosis with wilting and even withering (example isotig15576). (E) Moderate necrosis (example isotig19600). (F) Aggravation of PVX symptoms (example isotig15773). (G) No obvious difference compared to the eGFP control (example isotig14561). (H) Stunting indicated by a significant decrease in average plant height after infiltration with isotig18549, isotig13069, isotig18925, or isotig19369 compared to the eGFP control ( P
Figure Legend Snippet: Symptoms of systemic transient expression of Heterodera avenae effectors in Nicotiana benthamiana . (A) Untreated wild plant. (B) Empty vector control. (C) eGFP control. (D) Severe necrosis with wilting and even withering (example isotig15576). (E) Moderate necrosis (example isotig19600). (F) Aggravation of PVX symptoms (example isotig15773). (G) No obvious difference compared to the eGFP control (example isotig14561). (H) Stunting indicated by a significant decrease in average plant height after infiltration with isotig18549, isotig13069, isotig18925, or isotig19369 compared to the eGFP control ( P

Techniques Used: Expressing, Plasmid Preparation

RNA-silencing suppression assay of candidate Heterodera avenae effectors in Nicotiana benthamiana (example isotig18549). (A) Negative control: N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens cells containing the empty pGD vector and pGD-eGFP showing no green fluorescence. (B) Positive control: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-p19 and pGD-eGFP showing green fluorescence. (C) Example isotig18549: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-isotig18549 and pGD-eGFP showing no green fluorescence.
Figure Legend Snippet: RNA-silencing suppression assay of candidate Heterodera avenae effectors in Nicotiana benthamiana (example isotig18549). (A) Negative control: N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens cells containing the empty pGD vector and pGD-eGFP showing no green fluorescence. (B) Positive control: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-p19 and pGD-eGFP showing green fluorescence. (C) Example isotig18549: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-isotig18549 and pGD-eGFP showing no green fluorescence.

Techniques Used: Suppression Assay, Negative Control, Plasmid Preparation, Fluorescence, Positive Control

Effect of Heterodera avenae candidate effectors on Nicotiana benthamiana PCD. (A) Number and proportion of putative effector genes that induce PCD, suppress BAX-triggered cell death (BT-PCD) or have no effect on leaves of N. benthamiana . (B) Putative effectors that trigger cell death and chlorosis symptoms in N. benthamiana compared to eGFP as the negative control. (C) Suppression of BT-PCD in N. benthamiana by effectors (example isotig18549). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying isotig18549 or the negative control eGFP gene; infiltration was either performed alone or followed 24 h later by infiltration with A. tumefaciens cells carrying a mouse Bax gene. Western blotting confirmed the expression of BAX. (D) Necrosis indices of the infiltration spots of the example gene isotig18549 and control eGFP followed by Bax . Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of isotig18549 compared with that of eGFP ( P
Figure Legend Snippet: Effect of Heterodera avenae candidate effectors on Nicotiana benthamiana PCD. (A) Number and proportion of putative effector genes that induce PCD, suppress BAX-triggered cell death (BT-PCD) or have no effect on leaves of N. benthamiana . (B) Putative effectors that trigger cell death and chlorosis symptoms in N. benthamiana compared to eGFP as the negative control. (C) Suppression of BT-PCD in N. benthamiana by effectors (example isotig18549). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying isotig18549 or the negative control eGFP gene; infiltration was either performed alone or followed 24 h later by infiltration with A. tumefaciens cells carrying a mouse Bax gene. Western blotting confirmed the expression of BAX. (D) Necrosis indices of the infiltration spots of the example gene isotig18549 and control eGFP followed by Bax . Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of isotig18549 compared with that of eGFP ( P

Techniques Used: Negative Control, Western Blot, Expressing, Standard Deviation

Assay of the suppression of PTI (triggered by psojNIP) and ETI (triggered by Avr3a/R3a or Rbp-1/Gpa2) by Heterodera avenae candidate effectors in Nicotiana benthamiana . (A,C) Visualization of the phenotype of example isotig18549, which suppressed PTI triggered by psojNIP and ETI triggered by Avr3a/R3a. Western blotting confirmed the expression of psojNIP. (E) Visualization of the phenotypes of necrosis suppression (example isotig18549) and no suppression (example isotig15186) of ETI triggered by Rbp-1/Gpa2). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying the effector genes isotig18549 or isotig15186 or the negative control ( eGFP or empty vector PMD1) either alone or followed 24 h later by A. tumefaciens cells carrying the psojNIP, Avr3a / R3a or Rbp-1 / Gpa2 genes. (B,D,F) Necrosis indices of the infiltration spots of the 10 selected effector genes and controls ( eGFP or empty vector PMD1) followed by infiltration with vectors carrying the psojNIP , Avr3a / R3a or Rbp-1 / Gpa2 genes. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index compared with the control ( P
Figure Legend Snippet: Assay of the suppression of PTI (triggered by psojNIP) and ETI (triggered by Avr3a/R3a or Rbp-1/Gpa2) by Heterodera avenae candidate effectors in Nicotiana benthamiana . (A,C) Visualization of the phenotype of example isotig18549, which suppressed PTI triggered by psojNIP and ETI triggered by Avr3a/R3a. Western blotting confirmed the expression of psojNIP. (E) Visualization of the phenotypes of necrosis suppression (example isotig18549) and no suppression (example isotig15186) of ETI triggered by Rbp-1/Gpa2). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying the effector genes isotig18549 or isotig15186 or the negative control ( eGFP or empty vector PMD1) either alone or followed 24 h later by A. tumefaciens cells carrying the psojNIP, Avr3a / R3a or Rbp-1 / Gpa2 genes. (B,D,F) Necrosis indices of the infiltration spots of the 10 selected effector genes and controls ( eGFP or empty vector PMD1) followed by infiltration with vectors carrying the psojNIP , Avr3a / R3a or Rbp-1 / Gpa2 genes. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index compared with the control ( P

Techniques Used: Western Blot, Expressing, Negative Control, Plasmid Preparation, Standard Deviation

27) Product Images from "Developing cellulolytic Yarrowia lipolytica as a platform for the production of valuable products in consolidated bioprocessing of cellulose"

Article Title: Developing cellulolytic Yarrowia lipolytica as a platform for the production of valuable products in consolidated bioprocessing of cellulose

Journal: Biotechnology for Biofuels

doi: 10.1186/s13068-018-1144-6

Comparison of the growth, sugar consumption and lipase production of recombinant Y. lipolytica strains YLpW, YLpL (∆pox strain overexpressing LIP2 ) and and CYLpL (cellulolytic ∆pox strain overexpressing LIP2 ) during aerobic batch cultures on a YT and b YTD media versus time. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means
Figure Legend Snippet: Comparison of the growth, sugar consumption and lipase production of recombinant Y. lipolytica strains YLpW, YLpL (∆pox strain overexpressing LIP2 ) and and CYLpL (cellulolytic ∆pox strain overexpressing LIP2 ) during aerobic batch cultures on a YT and b YTD media versus time. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means

Techniques Used: Recombinant, Standard Deviation

Ricinoleic acid production of recombinant Y. lipolytica strains YLxR and CYLxR during aerobic batch culture in YTC media. a Comparison of growth and cellulose consumption and b ricinoleic acid production in 5 days. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means
Figure Legend Snippet: Ricinoleic acid production of recombinant Y. lipolytica strains YLxR and CYLxR during aerobic batch culture in YTC media. a Comparison of growth and cellulose consumption and b ricinoleic acid production in 5 days. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means

Techniques Used: Recombinant, Standard Deviation

Comparison of the growth, cellulose consumption and lipase production of recombinant Y. lipolytica strains a YLpL and b CYLpL during aerobic cultures on YTC media versus time. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means
Figure Legend Snippet: Comparison of the growth, cellulose consumption and lipase production of recombinant Y. lipolytica strains a YLpL and b CYLpL during aerobic cultures on YTC media versus time. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means

Techniques Used: Recombinant, Standard Deviation

Comparison of growth and glucose consumption of recombinant Y. lipolytica strains YLxW (prototrophic OleoX strain), YLxR (OleoX strain overexpressing CpFAH12 ) and CYLxR (cellulolytic OleoX strain overexpressing CpFAH12 ) during aerobic batch culture in a YT media and b YTD media versus time, and c ricinoleic acid production in 2 days. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means
Figure Legend Snippet: Comparison of growth and glucose consumption of recombinant Y. lipolytica strains YLxW (prototrophic OleoX strain), YLxR (OleoX strain overexpressing CpFAH12 ) and CYLxR (cellulolytic OleoX strain overexpressing CpFAH12 ) during aerobic batch culture in a YT media and b YTD media versus time, and c ricinoleic acid production in 2 days. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means

Techniques Used: Recombinant, Standard Deviation

Strategies used in the current study to develop a cellulolytic Y. lipolytica for CBP of cellulose. Products of interest are shown in color boxes
Figure Legend Snippet: Strategies used in the current study to develop a cellulolytic Y. lipolytica for CBP of cellulose. Products of interest are shown in color boxes

Techniques Used:

Comparison of lipid production by recombinant Y. lipolytica strains YLpW (prototrophic ∆pox strain), YLpO (∆pox strain overexpressing SCD1 and DGA1 ) and CYLpO (cellulolytic ∆pox strain overexpressing SCD1 and DGA1 ) during aerobic batch culture in minimum media supplemented with 80 g/L glucose under nitrogen starvation. Shown are a glucose consumption, b biomass formation and c FAME production versus time, and d the fatty acids profile. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means
Figure Legend Snippet: Comparison of lipid production by recombinant Y. lipolytica strains YLpW (prototrophic ∆pox strain), YLpO (∆pox strain overexpressing SCD1 and DGA1 ) and CYLpO (cellulolytic ∆pox strain overexpressing SCD1 and DGA1 ) during aerobic batch culture in minimum media supplemented with 80 g/L glucose under nitrogen starvation. Shown are a glucose consumption, b biomass formation and c FAME production versus time, and d the fatty acids profile. Values plotted at each time point are the means of three replications. Error bars represent standard deviation from the means

Techniques Used: Recombinant, Standard Deviation

Comparison of growth, cellulose consumption and lipid production of recombinant Y. lipolytica strains. a CYLpO, b CYLpO with the addition of 10 FPU cellulases/g cellulose, c YLpO with the addition of 20 FPU cellulases/g cellulose and d YLpO with the addition of 10 FPU cellulases/g cellulose during aerobic batch culture in minimum media containing 72.8 g/L cellulose
Figure Legend Snippet: Comparison of growth, cellulose consumption and lipid production of recombinant Y. lipolytica strains. a CYLpO, b CYLpO with the addition of 10 FPU cellulases/g cellulose, c YLpO with the addition of 20 FPU cellulases/g cellulose and d YLpO with the addition of 10 FPU cellulases/g cellulose during aerobic batch culture in minimum media containing 72.8 g/L cellulose

Techniques Used: Recombinant

28) Product Images from "Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells"

Article Title: Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18010040

GFP-tagged type 3 AR proteins localize to both the cytoplasm and nuclei of AR-negative prostate cancer PC3 cells under androgen-depleted conditions; co-expression with wild-type AR enhances the nuclear localization of type 3 AR in the presence of DHT. ( A ) Fluorescence images of PC3 cells transfected with GFP-tagged wild-type AR (GFP/AR WT), GFP/type 3a AR, GFP/type 3b AR, or co-transfection of untagged AR WT with GFP-tagged type 3a or 3b AR (green) and DAPI (blue) after 4 h treatment with vehicle or 10 nM DHT. Scale bars represent 20 µm; ( B ) Average mean fluorescence ratio of nuclear:cytoplasmic GFP/type 3a AR or GFP/type 3b AR in vehicle- or DHT-treated PC3 cells; ( C ) Average mean fluorescence ratio of nuclear:cytoplasmic GFP/AR WT in vehicle- or DHT-treated PC3 cells. Fluorescence from a total number of 20–35 cells per condition was quantified as described under Methods. Error bars represent SEM. Statistical analysis was carried out by GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) using two-way ANOVA ( B ) or independent t -test ( C ). p
Figure Legend Snippet: GFP-tagged type 3 AR proteins localize to both the cytoplasm and nuclei of AR-negative prostate cancer PC3 cells under androgen-depleted conditions; co-expression with wild-type AR enhances the nuclear localization of type 3 AR in the presence of DHT. ( A ) Fluorescence images of PC3 cells transfected with GFP-tagged wild-type AR (GFP/AR WT), GFP/type 3a AR, GFP/type 3b AR, or co-transfection of untagged AR WT with GFP-tagged type 3a or 3b AR (green) and DAPI (blue) after 4 h treatment with vehicle or 10 nM DHT. Scale bars represent 20 µm; ( B ) Average mean fluorescence ratio of nuclear:cytoplasmic GFP/type 3a AR or GFP/type 3b AR in vehicle- or DHT-treated PC3 cells; ( C ) Average mean fluorescence ratio of nuclear:cytoplasmic GFP/AR WT in vehicle- or DHT-treated PC3 cells. Fluorescence from a total number of 20–35 cells per condition was quantified as described under Methods. Error bars represent SEM. Statistical analysis was carried out by GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA) using two-way ANOVA ( B ) or independent t -test ( C ). p

Techniques Used: Expressing, Fluorescence, Transfection, Cotransfection, Software

29) Product Images from "Transcriptional responses of wheat and the cereal cyst nematode Heterodera avenae during their early contact stage"

Article Title: Transcriptional responses of wheat and the cereal cyst nematode Heterodera avenae during their early contact stage

Journal: Scientific Reports

doi: 10.1038/s41598-017-14047-y

Assay for suppression of BAX-triggered cell death (BT-PCD) by the candidate Heterodera avenae effectors ( a ) c68622.graph_c0 and ( b ) c72543.graph_c0 in Nicotiana benthamiana . Leaves of N. benthamiana were infiltrated with the infiltration buffer or Agrobacterium tumefaciens cells containing a pGR107 vector carrying the candidate effector gene either alone or infiltration with A. tumefaciens cells carrying a mouse Bax gene 24 h later. Photos of the phenotypes of infiltrated N. benthamiana leaves were taken 6 days after infiltration. The spots with label 3 on the leaf show that the gene c68622.graph_c0 suppressed the necrosis induced by Bax , but the gene c72543.graph_c0 did not suppress necrosis.
Figure Legend Snippet: Assay for suppression of BAX-triggered cell death (BT-PCD) by the candidate Heterodera avenae effectors ( a ) c68622.graph_c0 and ( b ) c72543.graph_c0 in Nicotiana benthamiana . Leaves of N. benthamiana were infiltrated with the infiltration buffer or Agrobacterium tumefaciens cells containing a pGR107 vector carrying the candidate effector gene either alone or infiltration with A. tumefaciens cells carrying a mouse Bax gene 24 h later. Photos of the phenotypes of infiltrated N. benthamiana leaves were taken 6 days after infiltration. The spots with label 3 on the leaf show that the gene c68622.graph_c0 suppressed the necrosis induced by Bax , but the gene c72543.graph_c0 did not suppress necrosis.

Techniques Used: Plasmid Preparation

30) Product Images from "Cloning and Functional Verification of Genes Related to 2-Phenylethanol Biosynthesis in Rosa rugosa"

Article Title: Cloning and Functional Verification of Genes Related to 2-Phenylethanol Biosynthesis in Rosa rugosa

Journal: Genes

doi: 10.3390/genes9120576

Analysis of the main volatile components in flower of RrPPDC1 transgenic and control Petunia . ( A ) methyl benzoate; ( B ) benzyl benzoate; ( C ) isoeugenol; ( D ) benzyl tiglate; ( E ) 2-phenylethanol; ( F ) eugenol; ( G ) benzyl alcohol; ( H ) benzoic acid, 2-phenylethyl ester; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (1–3) RrPPDC1 transgenic plants (±SE, n = 3).
Figure Legend Snippet: Analysis of the main volatile components in flower of RrPPDC1 transgenic and control Petunia . ( A ) methyl benzoate; ( B ) benzyl benzoate; ( C ) isoeugenol; ( D ) benzyl tiglate; ( E ) 2-phenylethanol; ( F ) eugenol; ( G ) benzyl alcohol; ( H ) benzoic acid, 2-phenylethyl ester; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (1–3) RrPPDC1 transgenic plants (±SE, n = 3).

Techniques Used: Transgenic Assay

Analysis of the main volatile components in flower of Petunia . (A1–H1) Main volatile components of RrAADC transgenic and control plants; (A2–H2) main volatile components of RrAAAT transgenic and control plants; ( A ) methyl benzoate; ( B ) benzyl benzoate; ( C ) isoeugenol; ( D ) benzyl tiglate; ( E ) 2-phenylethanol; ( F ) eugenol; ( G ) benzyl alcohol; ( H ) benzoic acid, 2-phenylethyl ester; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (1–3) transgenic plants. (±SE, n = 3).
Figure Legend Snippet: Analysis of the main volatile components in flower of Petunia . (A1–H1) Main volatile components of RrAADC transgenic and control plants; (A2–H2) main volatile components of RrAAAT transgenic and control plants; ( A ) methyl benzoate; ( B ) benzyl benzoate; ( C ) isoeugenol; ( D ) benzyl tiglate; ( E ) 2-phenylethanol; ( F ) eugenol; ( G ) benzyl alcohol; ( H ) benzoic acid, 2-phenylethyl ester; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (1–3) transgenic plants. (±SE, n = 3).

Techniques Used: Transgenic Assay

Phenotype and RT-PCR analysis of wild-type and RrPPDC1 transgenic Petunia . ( A , B ) Plant morphology of Petunia plants after 150 days of transplanting; ( C ) mRNA expression of RrPPDC1 in the flowers of transgenic and control Petunia plants; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (W) Water; (1–3) transgenic plants.
Figure Legend Snippet: Phenotype and RT-PCR analysis of wild-type and RrPPDC1 transgenic Petunia . ( A , B ) Plant morphology of Petunia plants after 150 days of transplanting; ( C ) mRNA expression of RrPPDC1 in the flowers of transgenic and control Petunia plants; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (W) Water; (1–3) transgenic plants.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Expressing

Phenotype and RT-PCR analysis of wild-type and RrAADC and RrAAAT transgenic Petunia . ( A1 , A2 ) Plant morphology of Petunia plant after 150 days of transplanting; ( B1 , B2 ) messenger RNA (mRNA) expression of RrAADC and RrAAAT in the flowers of transgenic Petunia plants; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (W) Water; (1–3) transgenic plants.
Figure Legend Snippet: Phenotype and RT-PCR analysis of wild-type and RrAADC and RrAAAT transgenic Petunia . ( A1 , A2 ) Plant morphology of Petunia plant after 150 days of transplanting; ( B1 , B2 ) messenger RNA (mRNA) expression of RrAADC and RrAAAT in the flowers of transgenic Petunia plants; (WT) wild type; (1304) pCAMBIA1304 transgenic plants; (W) Water; (1–3) transgenic plants.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Transgenic Assay, Expressing

31) Product Images from "The Impact of the Antigenic Composition of Chimeric Proteins on Their Immunoprotective Activity against Chronic Toxoplasmosis in Mice"

Article Title: The Impact of the Antigenic Composition of Chimeric Proteins on Their Immunoprotective Activity against Chronic Toxoplasmosis in Mice

Journal: Vaccines

doi: 10.3390/vaccines7040154

Western blot analysis of the tetravalent recombinant chimeric proteins. Recombinant proteins SAG1-MIC1-MAG1-GRA2 (SMMG; lane 1) and SAG2-GRA1-ROP1-GRA2 (SGRG; lane 2) were detected using specific anti-His antibodies and compared to the protein marker (M).
Figure Legend Snippet: Western blot analysis of the tetravalent recombinant chimeric proteins. Recombinant proteins SAG1-MIC1-MAG1-GRA2 (SMMG; lane 1) and SAG2-GRA1-ROP1-GRA2 (SGRG; lane 2) were detected using specific anti-His antibodies and compared to the protein marker (M).

Techniques Used: Western Blot, Recombinant, Marker

32) Product Images from "A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc03678d"

Article Title: A proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics proof-reading mechanism for non-proteinogenic amino acid incorporation into glycopeptide antibiotics †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9sc03678d

Journal: Chemical Science

doi: 10.1039/c9sc03678d

Reconstitution of peptide biosynthesis from the teicoplanin NRPS proteins Tcp10 and Tcp11, utilising two strategies to isolate individual modules 4–6 from Tcp11: either a C-A-PCP-E module architecture (A and B) or A-PCP-E-C architecture (C), together with the rationale behind the need for modularisation of the NRPS – the ability to load individual modules with peptide substrates using phosphopantetheinyl transferases (D). Rate of activation of the natural A-domain substrates for (A) and (C) were determined using a continuous, enzyme-coupled pyrophosphate detection assay; experiments performed in triplicate and standard deviation indicated. Peptide biosynthesis was reconstituted from tripeptide 3T loaded on M3, together with ATP, 4-Hpg and Tyr ( 1 ) using both the C-A-PCP-E module architecture and an M4–M5 fusion (B) or the A-PCP-E-C architecture (C). Peptide products were determined by LCMS analysis (ESI, positive mode), with solid lines indicating methylamide peptides (PCP-bound) and dashed lines indicating hydrolysed peptides (tripeptide 3T : black line; tetrapeptide 4T : dark grey line; pentapeptide 5T : light grey line; hexapeptide 6T-1 : blue line). A – adenylation domain, C – condensation domain, PCP – peptidyl carrier protein domain, E – epimerisation domain, Hpg – 4-hydroxyphenylglycine, Tyr – tyrosine ( 1 ).
Figure Legend Snippet: Reconstitution of peptide biosynthesis from the teicoplanin NRPS proteins Tcp10 and Tcp11, utilising two strategies to isolate individual modules 4–6 from Tcp11: either a C-A-PCP-E module architecture (A and B) or A-PCP-E-C architecture (C), together with the rationale behind the need for modularisation of the NRPS – the ability to load individual modules with peptide substrates using phosphopantetheinyl transferases (D). Rate of activation of the natural A-domain substrates for (A) and (C) were determined using a continuous, enzyme-coupled pyrophosphate detection assay; experiments performed in triplicate and standard deviation indicated. Peptide biosynthesis was reconstituted from tripeptide 3T loaded on M3, together with ATP, 4-Hpg and Tyr ( 1 ) using both the C-A-PCP-E module architecture and an M4–M5 fusion (B) or the A-PCP-E-C architecture (C). Peptide products were determined by LCMS analysis (ESI, positive mode), with solid lines indicating methylamide peptides (PCP-bound) and dashed lines indicating hydrolysed peptides (tripeptide 3T : black line; tetrapeptide 4T : dark grey line; pentapeptide 5T : light grey line; hexapeptide 6T-1 : blue line). A – adenylation domain, C – condensation domain, PCP – peptidyl carrier protein domain, E – epimerisation domain, Hpg – 4-hydroxyphenylglycine, Tyr – tyrosine ( 1 ).

Techniques Used: Activation Assay, Detection Assay, Standard Deviation, Liquid Chromatography with Mass Spectroscopy

33) Product Images from "Transcription factor TFAP2B up-regulates human corneal endothelial cell–specific genes during corneal development and maintenance"

Article Title: Transcription factor TFAP2B up-regulates human corneal endothelial cell–specific genes during corneal development and maintenance

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005527

Isolation of ZP4-expressing cells in CECs. A , cultured human CECs were separated into ZP4-negative or ZP4-positive ( ZP4 +) populations by FACS. B , expression of corneal endothelium–related genes in the sorted cells. ZP4-positive cells ( ZP4 +) highly expressed ZP4, TFAP2B, and COL8A2 compared with ZP4-negative cells ( ZP4 −). The expression levels were normalized to those of ZP4-negative cells. The cells collected by FACS were seeded at 20,000/per well in a 96-well plate and analyzed after 2 or 7 days. C , immunostaining of cells cultured for 7 days after cell sorting. Isolated ZP4-positive or ZP4-negative cells were cultured ( D ), and cell proliferation assays were performed after 2 days ( E ). Data are shown as the mean ± S.D. ( error bars ) ( n = 4). *, p
Figure Legend Snippet: Isolation of ZP4-expressing cells in CECs. A , cultured human CECs were separated into ZP4-negative or ZP4-positive ( ZP4 +) populations by FACS. B , expression of corneal endothelium–related genes in the sorted cells. ZP4-positive cells ( ZP4 +) highly expressed ZP4, TFAP2B, and COL8A2 compared with ZP4-negative cells ( ZP4 −). The expression levels were normalized to those of ZP4-negative cells. The cells collected by FACS were seeded at 20,000/per well in a 96-well plate and analyzed after 2 or 7 days. C , immunostaining of cells cultured for 7 days after cell sorting. Isolated ZP4-positive or ZP4-negative cells were cultured ( D ), and cell proliferation assays were performed after 2 days ( E ). Data are shown as the mean ± S.D. ( error bars ) ( n = 4). *, p

Techniques Used: Isolation, Expressing, Cell Culture, FACS, Immunostaining

Repression of TFAP2B in human cultured CECs. The siRNA-mediated knockdown in human cultured CECs was performed with control siRNA ( siControl ) and TFAP2B siRNA ( siTFAP2B ) for 48 h. A , real-time qRT-PCR of the neural crest marker SOX9; periocular mesenchyme markers PITX2, FOXC1, and FOXC2; and CEC markers TJP1 (ZO-1), Na + /K + -ATPase, COL8A1, COL8A2, and ZP4 following TFAP2B siRNA treatment. The expression levels were normalized to those of the siControl-treated CECs ( n = 4). B , Western blotting of TFAP2B siRNA–treated cultured CECs. C , immunofluorescence images of COL8A2 ( left panel , red ) and ZP4 ( right panel , green ) in human corneal sections. D , whole-mount immunofluorescence images of TFAP2B ( red ) and ZP4 ( green ). The TFAP2B-positive cells strongly expressed ZP4 protein in the human corneal endothelium. Hoechst 33342 ( blue ) was used to stain the nucleus. E , cell proliferation assay using AlamarBlue reagent of siControl- and siTFAP2B-treated CECs ( n = 6, duplicate three donors). The cells were seeded at 3,000 cells/well in a 96-well plate and analyzed after 2 days. The data are shown as the mean ± S.D. ( error bars ) *, p
Figure Legend Snippet: Repression of TFAP2B in human cultured CECs. The siRNA-mediated knockdown in human cultured CECs was performed with control siRNA ( siControl ) and TFAP2B siRNA ( siTFAP2B ) for 48 h. A , real-time qRT-PCR of the neural crest marker SOX9; periocular mesenchyme markers PITX2, FOXC1, and FOXC2; and CEC markers TJP1 (ZO-1), Na + /K + -ATPase, COL8A1, COL8A2, and ZP4 following TFAP2B siRNA treatment. The expression levels were normalized to those of the siControl-treated CECs ( n = 4). B , Western blotting of TFAP2B siRNA–treated cultured CECs. C , immunofluorescence images of COL8A2 ( left panel , red ) and ZP4 ( right panel , green ) in human corneal sections. D , whole-mount immunofluorescence images of TFAP2B ( red ) and ZP4 ( green ). The TFAP2B-positive cells strongly expressed ZP4 protein in the human corneal endothelium. Hoechst 33342 ( blue ) was used to stain the nucleus. E , cell proliferation assay using AlamarBlue reagent of siControl- and siTFAP2B-treated CECs ( n = 6, duplicate three donors). The cells were seeded at 3,000 cells/well in a 96-well plate and analyzed after 2 days. The data are shown as the mean ± S.D. ( error bars ) *, p

Techniques Used: Cell Culture, Quantitative RT-PCR, Marker, Capillary Electrochromatography, Expressing, Western Blot, Immunofluorescence, Staining, Proliferation Assay

Transcriptional activities of the COL8A2 and ZP4 promoters with TFAP2B. A and E , scheme of the luciferase reporter vectors of the human COL8A2 and ZP4 promoters. Mutations in the TFAP2B-binding site are shown in lowercase letters. B and F , EMSA of candidate TFAP2B-binding site of COL8A2 and ZP4 promoters. The shifted bands of the DNA–TFAP2B protein complexes were only observed in WT sequences of COL8A2 and ZP4. C and G , luciferase assays. TFAP2B-overexpressing 293T cells were transfected with the luciferase reporter vector containing the promoter regions of the COL8A2 and ZP4 genes. The luciferase activities were compared between the WT ( COL8A2 or ZP4 ) and TFAP2B-binding sequence mutant ( COL8A2 mutant and ZP4 mutant) luciferase vectors. The data were normalized to the luciferase activity of the WT. D and H , ChIP. DNA–protein complexes of cultured CECs were immunoprecipitated with IgG control or anti-TFAP2B antibody. Purified DNA was amplified using PCR and separated on an agarose gel. The input samples were used as a control. The data are shown as the mean ± S.D. ( error bars ) ( n = 4). *, p
Figure Legend Snippet: Transcriptional activities of the COL8A2 and ZP4 promoters with TFAP2B. A and E , scheme of the luciferase reporter vectors of the human COL8A2 and ZP4 promoters. Mutations in the TFAP2B-binding site are shown in lowercase letters. B and F , EMSA of candidate TFAP2B-binding site of COL8A2 and ZP4 promoters. The shifted bands of the DNA–TFAP2B protein complexes were only observed in WT sequences of COL8A2 and ZP4. C and G , luciferase assays. TFAP2B-overexpressing 293T cells were transfected with the luciferase reporter vector containing the promoter regions of the COL8A2 and ZP4 genes. The luciferase activities were compared between the WT ( COL8A2 or ZP4 ) and TFAP2B-binding sequence mutant ( COL8A2 mutant and ZP4 mutant) luciferase vectors. The data were normalized to the luciferase activity of the WT. D and H , ChIP. DNA–protein complexes of cultured CECs were immunoprecipitated with IgG control or anti-TFAP2B antibody. Purified DNA was amplified using PCR and separated on an agarose gel. The input samples were used as a control. The data are shown as the mean ± S.D. ( error bars ) ( n = 4). *, p

Techniques Used: Luciferase, Binding Assay, Transfection, Plasmid Preparation, Sequencing, Mutagenesis, Activity Assay, Chromatin Immunoprecipitation, Cell Culture, Immunoprecipitation, Purification, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

34) Product Images from "Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis"

Article Title: Installation of authentic BicA and SbtA proteins to the chloroplast envelope membrane is achieved by the proteolytic cleavage of chimeric proteins in Arabidopsis

Journal: Scientific Reports

doi: 10.1038/s41598-020-59190-1

Construct designs for the chimeric bicarbonate transporters and tobacco etch virus (TEV) protease. ( A ) Schematic diagram of the chimeric BicA and SbtA constructs used in this study. The protein A domain (pA) of the fusion constructs contains two IgG-binding domains from staphylococcal protein A. The human influenza hemagglutinin (HA) domain consists of the amino acids YPYDVPDYA. Both BicA and SbtA genes are derived from Synechocystis sp. PCC 6803. The K124 construct lacks the 6th transmembrane domain of Cor413im1. TP, the transit peptide of Cor413im1; TEV, TEV recognition sequence (ENLYFQG). ( B ) Schematic diagram of TEV protease. The TEV protease gene is derived from the tobacco etch virus. RBCS–TP represents the transit peptide of the small subunit of Ribluse-1,5-bisphosphate carboxylase/oxygenase (Rubisco). MBP, Maltose binding protein. ( C ) Prediction before and after transformation with the TEV protease construct. When the bicarbonate transporter chimeric protein was co-expressed with TEV protease, we predicted that the TEV recognition sequence is digested by TEV protease in the chloroplast.
Figure Legend Snippet: Construct designs for the chimeric bicarbonate transporters and tobacco etch virus (TEV) protease. ( A ) Schematic diagram of the chimeric BicA and SbtA constructs used in this study. The protein A domain (pA) of the fusion constructs contains two IgG-binding domains from staphylococcal protein A. The human influenza hemagglutinin (HA) domain consists of the amino acids YPYDVPDYA. Both BicA and SbtA genes are derived from Synechocystis sp. PCC 6803. The K124 construct lacks the 6th transmembrane domain of Cor413im1. TP, the transit peptide of Cor413im1; TEV, TEV recognition sequence (ENLYFQG). ( B ) Schematic diagram of TEV protease. The TEV protease gene is derived from the tobacco etch virus. RBCS–TP represents the transit peptide of the small subunit of Ribluse-1,5-bisphosphate carboxylase/oxygenase (Rubisco). MBP, Maltose binding protein. ( C ) Prediction before and after transformation with the TEV protease construct. When the bicarbonate transporter chimeric protein was co-expressed with TEV protease, we predicted that the TEV recognition sequence is digested by TEV protease in the chloroplast.

Techniques Used: Construct, Binding Assay, Derivative Assay, Periodic Counter-current Chromatography, Sequencing, Transformation Assay

35) Product Images from "Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia"

Article Title: Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.02838

The whole-genome average nucleotide identity (ANI) analysis of 29 Leptotrichia genomes generated by FastANI. The genetic relatedness between two genomes was estimated by ANI analysis. ≥95% ANI, which generally indicates genomes belonging to the same species, was highlighted by square in black.
Figure Legend Snippet: The whole-genome average nucleotide identity (ANI) analysis of 29 Leptotrichia genomes generated by FastANI. The genetic relatedness between two genomes was estimated by ANI analysis. ≥95% ANI, which generally indicates genomes belonging to the same species, was highlighted by square in black.

Techniques Used: Generated

Whole genome SNP-based majority phylogenetic tree of 29 Leptotrichia strains generated by kSNA3.0. Horizontal branch lengths indicate changes per number of SNPs. Strains in boldface are the strains their whole genomes were determined in this study. It should be noted that this is a SNPs-based (present in at least 75% strains), not an alignment based tree and no evolutionary direction can be inferred from this tree.
Figure Legend Snippet: Whole genome SNP-based majority phylogenetic tree of 29 Leptotrichia strains generated by kSNA3.0. Horizontal branch lengths indicate changes per number of SNPs. Strains in boldface are the strains their whole genomes were determined in this study. It should be noted that this is a SNPs-based (present in at least 75% strains), not an alignment based tree and no evolutionary direction can be inferred from this tree.

Techniques Used: Generated

Schematic representations of (A) Type I-B, (B) Type II-C, (C) Type III-A and -D, (D) Type III-like, and (E) Type VI-A of the CRISPR-Cas systems in Leptotrichia genomes. Genetic elements of CRISPR-Cas systems identified in this study are arranged according to their relative position in the chromosome. Different color arrows indicate different categories of cas genes: red, genes of cas8 , cas9 , cas10 , and cas13a ; blue, cas1 ; pink, cas3 ; yellow, cas4 ; orange, cas5 and csx1 ; cobalt blue, cas6 ; brown, cas7 and csx10 ; dark purple, csm2 ; purple, csm3 ; light yellow, csm4 ; light blue, csm5 ; deep green, csm6 ; gray, other genes. Left panels at the strain names represent phylogenetic parsimony trees calculated and drawn by kSNP3 and FigTree, respectively, based on DNA sequences covering all CRISPR/cas loci. Note that the regions with homologies of 64% or more show in gray bar.
Figure Legend Snippet: Schematic representations of (A) Type I-B, (B) Type II-C, (C) Type III-A and -D, (D) Type III-like, and (E) Type VI-A of the CRISPR-Cas systems in Leptotrichia genomes. Genetic elements of CRISPR-Cas systems identified in this study are arranged according to their relative position in the chromosome. Different color arrows indicate different categories of cas genes: red, genes of cas8 , cas9 , cas10 , and cas13a ; blue, cas1 ; pink, cas3 ; yellow, cas4 ; orange, cas5 and csx1 ; cobalt blue, cas6 ; brown, cas7 and csx10 ; dark purple, csm2 ; purple, csm3 ; light yellow, csm4 ; light blue, csm5 ; deep green, csm6 ; gray, other genes. Left panels at the strain names represent phylogenetic parsimony trees calculated and drawn by kSNP3 and FigTree, respectively, based on DNA sequences covering all CRISPR/cas loci. Note that the regions with homologies of 64% or more show in gray bar.

Techniques Used: CRISPR

Match of Leptotrichia CRISPR-Cas spacer sequences with sequences of phages, plasmids and bacterial genomes of own bacterial genus of Leptotrichia . This heat map shows number of spacers and the present of matched sequence with homology at least 90% in genomes of genus Leptotrichia . The first left panel indicates a total numbers of spacers present in each strain, and the second left panel does the numbers of spacer which have hit matches against each strain.
Figure Legend Snippet: Match of Leptotrichia CRISPR-Cas spacer sequences with sequences of phages, plasmids and bacterial genomes of own bacterial genus of Leptotrichia . This heat map shows number of spacers and the present of matched sequence with homology at least 90% in genomes of genus Leptotrichia . The first left panel indicates a total numbers of spacers present in each strain, and the second left panel does the numbers of spacer which have hit matches against each strain.

Techniques Used: CRISPR, Sequencing

36) Product Images from "Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix) Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix"

Article Title: Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix) Concerted functions of Streptococcus gordonii surface proteins PadA and Hsa mediate activation of human platelets and interactions with extracellular matrix

Journal: Cellular Microbiology

doi: 10.1111/cmi.12667

S. gordonii strains adherence to salivary pellicle and biofilm formation. Cover slips were coated with salivary pellicle and incubated with streptococcal cells (5 × 10 7 per well) for 2 hr at 37°C for adherence (grey columns) or in YPT‐Glc medium for 16 hr at 37°C for biofilm formation (black columns). Bacterial cells adhered, and biofilm biomass values were quantified by staining with crystal violet as described in 4 . Expression of PadA or Hsa proteins by complemented strains was induced with 10 ng or 50 ng nisin ml −1 , respectively. Error bars represent ±SEM from three independent experiments ( n = 3). * P
Figure Legend Snippet: S. gordonii strains adherence to salivary pellicle and biofilm formation. Cover slips were coated with salivary pellicle and incubated with streptococcal cells (5 × 10 7 per well) for 2 hr at 37°C for adherence (grey columns) or in YPT‐Glc medium for 16 hr at 37°C for biofilm formation (black columns). Bacterial cells adhered, and biofilm biomass values were quantified by staining with crystal violet as described in 4 . Expression of PadA or Hsa proteins by complemented strains was induced with 10 ng or 50 ng nisin ml −1 , respectively. Error bars represent ±SEM from three independent experiments ( n = 3). * P

Techniques Used: Incubation, Gas Chromatography, Staining, Expressing

Expression of PadA and Hsa proteins by S. gordonii DL1 wild type, mutants and complemented mutants. (a) (composite), SDS‐PAGE gel, and corresponding Western immunoblot of PadA (~350 kDa) in cell wall proteins extracted from: (1) DL1; (2) UB2723 Δ padA ; (3) UB2724 Δ padA /pMSP‐ padA ; (4) UB2773 Δ padA Δ hsa ; and (5) UB2775 Δ padA Δ hsa /pMSP‐ padA. In lanes 3 and 5, protein expression was induced with 10 ng nisin ml −1 . (b) (composite), whole cell dot blots reacted with sWGA of: (1) DL1; (2) UB2029 Δ hsa ; (3) UB2773 Δ padA Δ hsa ; (4) UB2777 Δ padA Δ hsa /pMSP‐ hsa ; (5) UB2777 + 10 ng nisin ml −1 ; and (6) UB2777 + 50 ng nisin ml −1 . (c) Biotinylated sWGA binding to immobilized bacterial cells (2 × 10 7 per well) followed by HRP‐streptavidin to detect Hsa as described in 4 . Figures in parentheses indicate ng ml −1 nisin added to cultures to induce expression of Hsa in the complemented strain UB2777 Δ padA Δ hsa /pMSP‐ hsa. Error bars represent ±SEM from two independent experiments ( n = 2). * P
Figure Legend Snippet: Expression of PadA and Hsa proteins by S. gordonii DL1 wild type, mutants and complemented mutants. (a) (composite), SDS‐PAGE gel, and corresponding Western immunoblot of PadA (~350 kDa) in cell wall proteins extracted from: (1) DL1; (2) UB2723 Δ padA ; (3) UB2724 Δ padA /pMSP‐ padA ; (4) UB2773 Δ padA Δ hsa ; and (5) UB2775 Δ padA Δ hsa /pMSP‐ padA. In lanes 3 and 5, protein expression was induced with 10 ng nisin ml −1 . (b) (composite), whole cell dot blots reacted with sWGA of: (1) DL1; (2) UB2029 Δ hsa ; (3) UB2773 Δ padA Δ hsa ; (4) UB2777 Δ padA Δ hsa /pMSP‐ hsa ; (5) UB2777 + 10 ng nisin ml −1 ; and (6) UB2777 + 50 ng nisin ml −1 . (c) Biotinylated sWGA binding to immobilized bacterial cells (2 × 10 7 per well) followed by HRP‐streptavidin to detect Hsa as described in 4 . Figures in parentheses indicate ng ml −1 nisin added to cultures to induce expression of Hsa in the complemented strain UB2777 Δ padA Δ hsa /pMSP‐ hsa. Error bars represent ±SEM from two independent experiments ( n = 2). * P

Techniques Used: Expressing, SDS Page, Western Blot, Binding Assay

Diagrammatic representation of some of the processes involved in platelet activation by S. gordonii . Cell wall‐anchored proteins Hsa and PadA interact with platelet membrane integrins GPIb and α IIb β 3 (GPIIbIIIa). Hsa captures platelets under flow (rolling) by binding GPIb, and possibly also α IIb β 3 , and activates signalling cascades including FcγRIIa phosphorylation, leading to dense granule release (see Arman et al., 2014 ). PadA binds activated α IIb β 3 , thus amplifying signals leading to shape change, thrombin production, coagulation, and thrombus formation. Platelet activation by S. gordonii can occur in the absence of specific IgG. However, with IgG present, there is evidence for activation (phosphorylation) of spleen tyrosine kinase (Syk‐P) through FcγRIIa. Conserved streptococcal surface protein antigens such as antigen I/II proteins (e.g., SspA/B) may also be involved in the overall process (Kerrigan et al., 2007 ). Physiologically, collagen activates Syk through GPVI, which is closely associated with FcγRIIa (not shown). Fibrinogen engages GPIIbIIIa (α IIb β 3 ), which also associates with FcγRIIa. CD40L (otherwise known as CD154) is up‐regulated in the platelet cell membrane and binds CD40 + cells such as endothelial cells and neutrophils, while soluble (released) CD40L further activates platelets
Figure Legend Snippet: Diagrammatic representation of some of the processes involved in platelet activation by S. gordonii . Cell wall‐anchored proteins Hsa and PadA interact with platelet membrane integrins GPIb and α IIb β 3 (GPIIbIIIa). Hsa captures platelets under flow (rolling) by binding GPIb, and possibly also α IIb β 3 , and activates signalling cascades including FcγRIIa phosphorylation, leading to dense granule release (see Arman et al., 2014 ). PadA binds activated α IIb β 3 , thus amplifying signals leading to shape change, thrombin production, coagulation, and thrombus formation. Platelet activation by S. gordonii can occur in the absence of specific IgG. However, with IgG present, there is evidence for activation (phosphorylation) of spleen tyrosine kinase (Syk‐P) through FcγRIIa. Conserved streptococcal surface protein antigens such as antigen I/II proteins (e.g., SspA/B) may also be involved in the overall process (Kerrigan et al., 2007 ). Physiologically, collagen activates Syk through GPVI, which is closely associated with FcγRIIa (not shown). Fibrinogen engages GPIIbIIIa (α IIb β 3 ), which also associates with FcγRIIa. CD40L (otherwise known as CD154) is up‐regulated in the platelet cell membrane and binds CD40 + cells such as endothelial cells and neutrophils, while soluble (released) CD40L further activates platelets

Techniques Used: Activation Assay, Flow Cytometry, Binding Assay, Coagulation

Adhesion of S. gordonii strains to human cellular fibronectin (cFn). Microtitre plate wells were coated with cFn (1 μg per well), blocked with BSA, and then incubated with streptococcal cells (5 × 10 7 per well) for 2 hr at 37°C. Bacterial cells adhered (black columns) were quantified by staining with crystal violet as described in 4 . Wells coated with cFn were also incubated with 0.001 U neuraminidase (sialidase) for 2 hr at 37°C, washed, blocked with BSA and then incubated with streptococcal cells (grey shaded columns). Expression of PadA or Hsa proteins by complemented strains was induced with 10 ng or 50 ng nisin ml −1 . Error bars represent ±SEM from three independent experiments ( n = 3). * P
Figure Legend Snippet: Adhesion of S. gordonii strains to human cellular fibronectin (cFn). Microtitre plate wells were coated with cFn (1 μg per well), blocked with BSA, and then incubated with streptococcal cells (5 × 10 7 per well) for 2 hr at 37°C. Bacterial cells adhered (black columns) were quantified by staining with crystal violet as described in 4 . Wells coated with cFn were also incubated with 0.001 U neuraminidase (sialidase) for 2 hr at 37°C, washed, blocked with BSA and then incubated with streptococcal cells (grey shaded columns). Expression of PadA or Hsa proteins by complemented strains was induced with 10 ng or 50 ng nisin ml −1 . Error bars represent ±SEM from three independent experiments ( n = 3). * P

Techniques Used: Incubation, Staining, Expressing

Adhesion of S. gordonii strains to human vitronectin (Vn). Microtitre plate wells were coated with Vn (0.05 μg per well), blocked with BSA, and then incubated with streptococcal cells (5 × 10 7 per well) for 2 hr at 37°C. Bacterial cells adhered (black columns) were quantified by staining with crystal violet as described in 4 . Wells coated with Vn were also incubated with 0.001 U neuraminidase (sialidase) for 2 hr at 37°C, washed, blocked with BSA, and then incubated with streptococcal cells (grey columns). Expression of PadA or Hsa proteins by complemented strains was induced with 10 ng or 50 ng nisin ml −1 , respectively. Error bars represent ±SEM from three independent experiments ( n = 3). * P
Figure Legend Snippet: Adhesion of S. gordonii strains to human vitronectin (Vn). Microtitre plate wells were coated with Vn (0.05 μg per well), blocked with BSA, and then incubated with streptococcal cells (5 × 10 7 per well) for 2 hr at 37°C. Bacterial cells adhered (black columns) were quantified by staining with crystal violet as described in 4 . Wells coated with Vn were also incubated with 0.001 U neuraminidase (sialidase) for 2 hr at 37°C, washed, blocked with BSA, and then incubated with streptococcal cells (grey columns). Expression of PadA or Hsa proteins by complemented strains was induced with 10 ng or 50 ng nisin ml −1 , respectively. Error bars represent ±SEM from three independent experiments ( n = 3). * P

Techniques Used: Incubation, Staining, Expressing

Platelet adhesion to immobilized S. gordonii strains under static conditions. Bacterial cells were deposited onto microwells, and platelet binding was determined by phosphatase assay as described in 4 . Figures in parentheses indicate ng ml −1 nisin added to cultures to induce expression of PadA or Hsa in the complemented strains UB2775 Δ padA Δ hsa /pMSP‐ padA , and UB2777 Δ padA Δ hsa /pMSP‐ hsa . Fibrinogen 100 μg ml −1 positive control; BSA 100 μg ml −1 negative control. Error bars represent ±SEM from four independent experiments ( n = 4). * P
Figure Legend Snippet: Platelet adhesion to immobilized S. gordonii strains under static conditions. Bacterial cells were deposited onto microwells, and platelet binding was determined by phosphatase assay as described in 4 . Figures in parentheses indicate ng ml −1 nisin added to cultures to induce expression of PadA or Hsa in the complemented strains UB2775 Δ padA Δ hsa /pMSP‐ padA , and UB2777 Δ padA Δ hsa /pMSP‐ hsa . Fibrinogen 100 μg ml −1 positive control; BSA 100 μg ml −1 negative control. Error bars represent ±SEM from four independent experiments ( n = 4). * P

Techniques Used: Binding Assay, Phosphatase Assay, Expressing, Positive Control, Negative Control

37) Product Images from "di-Cysteine motifs in the C-terminus of plant HMA4 proteins confer nanomolar affinity for zinc and are essential for HMA4 function in vivo"

Article Title: di-Cysteine motifs in the C-terminus of plant HMA4 proteins confer nanomolar affinity for zinc and are essential for HMA4 function in vivo

Journal: Journal of Experimental Botany

doi: 10.1093/jxb/ery311

Complementation of the A. thaliana hma2hma4 zinc deficiency phenotype. HMA4 variants were expressed in hma2hma4 plants under the control of the AtHMA4 promoter. The plant phenotypes are shown after 6 weeks of growth on soil without zinc supplementation. Non-transformed hma2hma4 plants or expressing the native HMA4 proteins were respectively used as negative and positive controls. Images are representative of multiple observations of four to eight independent homozygous T3 lines for each genotype. Ah, A. halleri ; At, A. thaliana ; AtAhHMA4 and AhAtHMA4, swapped C-terminal extensions; HA: His- → Ala-stretch; CCAA: di-Cys → di-Ala motifs; CHA: HA and CCAA mutations combined; Ctrunc: fully truncated C-terminal extension.
Figure Legend Snippet: Complementation of the A. thaliana hma2hma4 zinc deficiency phenotype. HMA4 variants were expressed in hma2hma4 plants under the control of the AtHMA4 promoter. The plant phenotypes are shown after 6 weeks of growth on soil without zinc supplementation. Non-transformed hma2hma4 plants or expressing the native HMA4 proteins were respectively used as negative and positive controls. Images are representative of multiple observations of four to eight independent homozygous T3 lines for each genotype. Ah, A. halleri ; At, A. thaliana ; AtAhHMA4 and AhAtHMA4, swapped C-terminal extensions; HA: His- → Ala-stretch; CCAA: di-Cys → di-Ala motifs; CHA: HA and CCAA mutations combined; Ctrunc: fully truncated C-terminal extension.

Techniques Used: Transformation Assay, Expressing

Zinc accumulation in complemented plants grown on soil. Non-transformed hma2hma4 mutant (white) and expressing the native or mutant A. thaliana (dark gray) or A. halleri (light gray) HMA4 proteins under the control of the AtHMA4 promoter were grown for 6 weeks on soil without zinc supplementation. Zinc concentrations were measured in shoot tissues collected from two plants per line. Values are relative to lines expressing the native AtHMA4 proteins and are means±SEM of four to eight homozygous T3 independent lines for each genotype. The data were analysed with one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences ( P
Figure Legend Snippet: Zinc accumulation in complemented plants grown on soil. Non-transformed hma2hma4 mutant (white) and expressing the native or mutant A. thaliana (dark gray) or A. halleri (light gray) HMA4 proteins under the control of the AtHMA4 promoter were grown for 6 weeks on soil without zinc supplementation. Zinc concentrations were measured in shoot tissues collected from two plants per line. Values are relative to lines expressing the native AtHMA4 proteins and are means±SEM of four to eight homozygous T3 independent lines for each genotype. The data were analysed with one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences ( P

Techniques Used: Transformation Assay, Mutagenesis, Expressing

Zinc accumulation and expression of zinc deficiency response genes in complemented plants grown in hydroponic conditions. Non-transformed hma2hma4 mutant (white) and expressing the native or mutant A. halleri HMA4 proteins (medium gray) under the control of AtHMA4 promoter were grown for the last 3 weeks before harvest in Hoagland hydroponic medium containing 0.2 µM zinc. Zinc concentrations were measured in shoot (A) and root (B) tissues collected from two plants per line. Values are relative to lines expressing the native AhHMA4 proteins and are means±SEM of two independent lines from three biological replicates. Transcript levels were quantified from plant tissues collected from two plants per line. Relative transcript levels (RTL) of IRT3 (C) and ZIP9 (D) in shoots are mean±SEM of two independent lines from two biological replicates. The data were analysed with one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences ( P
Figure Legend Snippet: Zinc accumulation and expression of zinc deficiency response genes in complemented plants grown in hydroponic conditions. Non-transformed hma2hma4 mutant (white) and expressing the native or mutant A. halleri HMA4 proteins (medium gray) under the control of AtHMA4 promoter were grown for the last 3 weeks before harvest in Hoagland hydroponic medium containing 0.2 µM zinc. Zinc concentrations were measured in shoot (A) and root (B) tissues collected from two plants per line. Values are relative to lines expressing the native AhHMA4 proteins and are means±SEM of two independent lines from three biological replicates. Transcript levels were quantified from plant tissues collected from two plants per line. Relative transcript levels (RTL) of IRT3 (C) and ZIP9 (D) in shoots are mean±SEM of two independent lines from two biological replicates. The data were analysed with one-way ANOVA followed by Tukey’s multiple comparison test. Statistically significant differences ( P

Techniques Used: Expressing, Transformation Assay, Mutagenesis

38) Product Images from "Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana"

Article Title: Large-Scale Identification and Characterization of Heterodera avenae Putative Effectors Suppressing or Inducing Cell Death in Nicotiana benthamiana

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2017.02062

Candidate Heterodera avenae effectors (example isotig18549) suppress cell death triggered by other candidate H. avenae effectors (example isotig12969) in Nicotiana benthamiana . (A) Assay of the suppression of isotig12969-triggered cell death in N. benthamiana by isotig18549. The results of the verification of gene expression of isotig18549 and isotig12969 by western blotting are shown below. (B) Necrosis index of isotig18549 and control eGFP followed by isotig12969. Each column shows the mean and standard deviation.
Figure Legend Snippet: Candidate Heterodera avenae effectors (example isotig18549) suppress cell death triggered by other candidate H. avenae effectors (example isotig12969) in Nicotiana benthamiana . (A) Assay of the suppression of isotig12969-triggered cell death in N. benthamiana by isotig18549. The results of the verification of gene expression of isotig18549 and isotig12969 by western blotting are shown below. (B) Necrosis index of isotig18549 and control eGFP followed by isotig12969. Each column shows the mean and standard deviation.

Techniques Used: Expressing, Western Blot, Standard Deviation

Symptoms of systemic transient expression of Heterodera avenae effectors in Nicotiana benthamiana . (A) Untreated wild plant. (B) Empty vector control. (C) eGFP control. (D) Severe necrosis with wilting and even withering (example isotig15576). (E) Moderate necrosis (example isotig19600). (F) Aggravation of PVX symptoms (example isotig15773). (G) No obvious difference compared to the eGFP control (example isotig14561). (H) Stunting indicated by a significant decrease in average plant height after infiltration with isotig18549, isotig13069, isotig18925, or isotig19369 compared to the eGFP control ( P
Figure Legend Snippet: Symptoms of systemic transient expression of Heterodera avenae effectors in Nicotiana benthamiana . (A) Untreated wild plant. (B) Empty vector control. (C) eGFP control. (D) Severe necrosis with wilting and even withering (example isotig15576). (E) Moderate necrosis (example isotig19600). (F) Aggravation of PVX symptoms (example isotig15773). (G) No obvious difference compared to the eGFP control (example isotig14561). (H) Stunting indicated by a significant decrease in average plant height after infiltration with isotig18549, isotig13069, isotig18925, or isotig19369 compared to the eGFP control ( P

Techniques Used: Expressing, Plasmid Preparation

RNA-silencing suppression assay of candidate Heterodera avenae effectors in Nicotiana benthamiana (example isotig18549). (A) Negative control: N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens cells containing the empty pGD vector and pGD-eGFP showing no green fluorescence. (B) Positive control: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-p19 and pGD-eGFP showing green fluorescence. (C) Example isotig18549: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-isotig18549 and pGD-eGFP showing no green fluorescence.
Figure Legend Snippet: RNA-silencing suppression assay of candidate Heterodera avenae effectors in Nicotiana benthamiana (example isotig18549). (A) Negative control: N. benthamiana leaves were infiltrated with a mixture of Agrobacterium tumefaciens cells containing the empty pGD vector and pGD-eGFP showing no green fluorescence. (B) Positive control: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-p19 and pGD-eGFP showing green fluorescence. (C) Example isotig18549: N. benthamiana leaves were infiltrated with a mixture of A. tumefaciens cells containing pGD-isotig18549 and pGD-eGFP showing no green fluorescence.

Techniques Used: Suppression Assay, Negative Control, Plasmid Preparation, Fluorescence, Positive Control

Effect of Heterodera avenae candidate effectors on Nicotiana benthamiana PCD. (A) Number and proportion of putative effector genes that induce PCD, suppress BAX-triggered cell death (BT-PCD) or have no effect on leaves of N. benthamiana . (B) Putative effectors that trigger cell death and chlorosis symptoms in N. benthamiana compared to eGFP as the negative control. (C) Suppression of BT-PCD in N. benthamiana by effectors (example isotig18549). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying isotig18549 or the negative control eGFP gene; infiltration was either performed alone or followed 24 h later by infiltration with A. tumefaciens cells carrying a mouse Bax gene. Western blotting confirmed the expression of BAX. (D) Necrosis indices of the infiltration spots of the example gene isotig18549 and control eGFP followed by Bax . Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of isotig18549 compared with that of eGFP ( P
Figure Legend Snippet: Effect of Heterodera avenae candidate effectors on Nicotiana benthamiana PCD. (A) Number and proportion of putative effector genes that induce PCD, suppress BAX-triggered cell death (BT-PCD) or have no effect on leaves of N. benthamiana . (B) Putative effectors that trigger cell death and chlorosis symptoms in N. benthamiana compared to eGFP as the negative control. (C) Suppression of BT-PCD in N. benthamiana by effectors (example isotig18549). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying isotig18549 or the negative control eGFP gene; infiltration was either performed alone or followed 24 h later by infiltration with A. tumefaciens cells carrying a mouse Bax gene. Western blotting confirmed the expression of BAX. (D) Necrosis indices of the infiltration spots of the example gene isotig18549 and control eGFP followed by Bax . Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index of isotig18549 compared with that of eGFP ( P

Techniques Used: Negative Control, Western Blot, Expressing, Standard Deviation

Assay of the suppression of PTI (triggered by psojNIP) and ETI (triggered by Avr3a/R3a or Rbp-1/Gpa2) by Heterodera avenae candidate effectors in Nicotiana benthamiana . (A,C) Visualization of the phenotype of example isotig18549, which suppressed PTI triggered by psojNIP and ETI triggered by Avr3a/R3a. Western blotting confirmed the expression of psojNIP. (E) Visualization of the phenotypes of necrosis suppression (example isotig18549) and no suppression (example isotig15186) of ETI triggered by Rbp-1/Gpa2). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying the effector genes isotig18549 or isotig15186 or the negative control ( eGFP or empty vector PMD1) either alone or followed 24 h later by A. tumefaciens cells carrying the psojNIP, Avr3a / R3a or Rbp-1 / Gpa2 genes. (B,D,F) Necrosis indices of the infiltration spots of the 10 selected effector genes and controls ( eGFP or empty vector PMD1) followed by infiltration with vectors carrying the psojNIP , Avr3a / R3a or Rbp-1 / Gpa2 genes. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index compared with the control ( P
Figure Legend Snippet: Assay of the suppression of PTI (triggered by psojNIP) and ETI (triggered by Avr3a/R3a or Rbp-1/Gpa2) by Heterodera avenae candidate effectors in Nicotiana benthamiana . (A,C) Visualization of the phenotype of example isotig18549, which suppressed PTI triggered by psojNIP and ETI triggered by Avr3a/R3a. Western blotting confirmed the expression of psojNIP. (E) Visualization of the phenotypes of necrosis suppression (example isotig18549) and no suppression (example isotig15186) of ETI triggered by Rbp-1/Gpa2). N. benthamiana leaves were infiltrated with buffer or with Agrobacterium tumefaciens cells carrying the effector genes isotig18549 or isotig15186 or the negative control ( eGFP or empty vector PMD1) either alone or followed 24 h later by A. tumefaciens cells carrying the psojNIP, Avr3a / R3a or Rbp-1 / Gpa2 genes. (B,D,F) Necrosis indices of the infiltration spots of the 10 selected effector genes and controls ( eGFP or empty vector PMD1) followed by infiltration with vectors carrying the psojNIP , Avr3a / R3a or Rbp-1 / Gpa2 genes. Each column shows the mean and standard deviation. The columns with asterisks show a statistically significant reduction of the necrosis index compared with the control ( P

Techniques Used: Western Blot, Expressing, Negative Control, Plasmid Preparation, Standard Deviation

39) Product Images from "MYC-type transcription factors, MYC67 and MYC70, interact with ICE1 and negatively regulate cold tolerance in Arabidopsis"

Article Title: MYC-type transcription factors, MYC67 and MYC70, interact with ICE1 and negatively regulate cold tolerance in Arabidopsis

Journal: Scientific Reports

doi: 10.1038/s41598-018-29722-x

MYC67 and MYC70 negatively regulate the expression of cold-responsive genes. ( A ) The relative mRNA transcript levels of CBF / DREB1 genes, COR47 , COR15A , KIN1 , and P5CS2 in wild-type, myc67 and myc70 seedlings were determined by quantitative RT-PCR analysis. Ten-day-old seedlings grown at 23 °C were incubated at 4 °C for the indicated amounts of time. The data indicate the means and SD (n = 3). ( B ) The relative mRNA transcript levels in wild-type, MYC67 -, and MYC70 -overexpressing seedlings were determined by quantitative RT-PCR. The data indicate the means and SD (n = 3). ( C ) The expression levels of MYC67 and MYC70 after cold treatment were measured by quantitative RT-PCR analysis. ( D ) Expression level of MYC67 and MYC70 after cold treatment in wild type and the ice1 - D mutant. Asterisks indicates a significant difference from wild-type or plants with a vector at each point (p
Figure Legend Snippet: MYC67 and MYC70 negatively regulate the expression of cold-responsive genes. ( A ) The relative mRNA transcript levels of CBF / DREB1 genes, COR47 , COR15A , KIN1 , and P5CS2 in wild-type, myc67 and myc70 seedlings were determined by quantitative RT-PCR analysis. Ten-day-old seedlings grown at 23 °C were incubated at 4 °C for the indicated amounts of time. The data indicate the means and SD (n = 3). ( B ) The relative mRNA transcript levels in wild-type, MYC67 -, and MYC70 -overexpressing seedlings were determined by quantitative RT-PCR. The data indicate the means and SD (n = 3). ( C ) The expression levels of MYC67 and MYC70 after cold treatment were measured by quantitative RT-PCR analysis. ( D ) Expression level of MYC67 and MYC70 after cold treatment in wild type and the ice1 - D mutant. Asterisks indicates a significant difference from wild-type or plants with a vector at each point (p

Techniques Used: Expressing, Quantitative RT-PCR, Incubation, Mutagenesis, Plasmid Preparation

Interactions between ICE1, MYC67, and MYC70. ( A ) A schematic representation of the deletion constructs for ICE1. The indicated regions of ICE1 were cloned into the bait vector pGBKT7. ( B ) The interaction of ICE1 with MYC67 or MYC70 was assessed using the yeast 2-hybrid assay. The ICE1 bait plasmid was transformed with either pACT2 (vector control), pACT2-MYC67 m2 (amino acids 3–358), or pACT2-MYC70 m4 (amino acids 246–359). Yeast grown on SD media without leucine and tryptophan (upper, −LW) and on selection plates (lower, SP) are shown. ( C ) MYC67 and MYC70 form homodimers and interact with each other. Yeast strains containing full-length pGBKT7-MYC67 or pGBKT7-MYC70 as bait were transformed with pACT2 (vector control), full-length pACT2-MYC67 or full-length pACT2-MYC70 as prey. Yeast was incubated on −LW or on SP. ( D ) Only ICE1 was able to interact with the C-terminal region of MYC70 in the yeast 2-hybrid assay. MYC70 full was interacted with MYC67, MYC70, and ICE1, but MYC70 m4 was able to interact with ICE1. ( E ) The effect of ICE1 point mutations, S403A or K393R, on the interactions with MYC67 and MYC70.
Figure Legend Snippet: Interactions between ICE1, MYC67, and MYC70. ( A ) A schematic representation of the deletion constructs for ICE1. The indicated regions of ICE1 were cloned into the bait vector pGBKT7. ( B ) The interaction of ICE1 with MYC67 or MYC70 was assessed using the yeast 2-hybrid assay. The ICE1 bait plasmid was transformed with either pACT2 (vector control), pACT2-MYC67 m2 (amino acids 3–358), or pACT2-MYC70 m4 (amino acids 246–359). Yeast grown on SD media without leucine and tryptophan (upper, −LW) and on selection plates (lower, SP) are shown. ( C ) MYC67 and MYC70 form homodimers and interact with each other. Yeast strains containing full-length pGBKT7-MYC67 or pGBKT7-MYC70 as bait were transformed with pACT2 (vector control), full-length pACT2-MYC67 or full-length pACT2-MYC70 as prey. Yeast was incubated on −LW or on SP. ( D ) Only ICE1 was able to interact with the C-terminal region of MYC70 in the yeast 2-hybrid assay. MYC70 full was interacted with MYC67, MYC70, and ICE1, but MYC70 m4 was able to interact with ICE1. ( E ) The effect of ICE1 point mutations, S403A or K393R, on the interactions with MYC67 and MYC70.

Techniques Used: Construct, Clone Assay, Plasmid Preparation, Y2H Assay, Transformation Assay, Selection, Incubation

MYC67 and MYC70 negatively regulate freezing tolerance. ( A ) The freezing tolerance of myc67 and myc70 after cold acclimation. Representative wild-type (i), myc67 (ii) and myc70 (iii) plants that were treated with −7 °C are shown. ( B ) Quantification of the survival rates for the cold-acclimated wild-type, myc67 , and myc70 plants after a −7 °C freezing treatment. The data indicate the means and standard errors (n = 6). The tolerance of myc67 , myc70 , and myc67 myc70 mutants was significantly different from that of wild-type plants (t-test, P
Figure Legend Snippet: MYC67 and MYC70 negatively regulate freezing tolerance. ( A ) The freezing tolerance of myc67 and myc70 after cold acclimation. Representative wild-type (i), myc67 (ii) and myc70 (iii) plants that were treated with −7 °C are shown. ( B ) Quantification of the survival rates for the cold-acclimated wild-type, myc67 , and myc70 plants after a −7 °C freezing treatment. The data indicate the means and standard errors (n = 6). The tolerance of myc67 , myc70 , and myc67 myc70 mutants was significantly different from that of wild-type plants (t-test, P

Techniques Used: T-Test

Expression and localization of MYC67 and MYC70 . ( A ) Six-week-old plants expressing MYC67pro:GUS was treated without (i–iii) or with (iv–vi) low temperature for 6 h. MYC67pro:GUS expression in mature rosetta leaves (i,iv), base of rosetta leaves (ii, v), and floral organs (iii,vi). ( B ) Six-week-old plants expressing MYC70pro:GUS was treated without (i–iii) or with (iv–vi) low temperature for 6 h. MYC70pro:GUS expression in mature rosetta leaves (i, iv), base of rosetta leaves (ii,v), and floral organs (iii, vi). ( C ) Subcellular localization of the GFP-MYC proteins. The nuclei were stained with 20 mg/ml PI. The scale bar indicates 20 μm. ( D ) GFP fluorescence at the root tips. Seven-day old seedlings were treated at 4 °C for 3 h, and GFP fluorescence was observed by confocal microscopy. The scale bar indicates 150 μm.
Figure Legend Snippet: Expression and localization of MYC67 and MYC70 . ( A ) Six-week-old plants expressing MYC67pro:GUS was treated without (i–iii) or with (iv–vi) low temperature for 6 h. MYC67pro:GUS expression in mature rosetta leaves (i,iv), base of rosetta leaves (ii, v), and floral organs (iii,vi). ( B ) Six-week-old plants expressing MYC70pro:GUS was treated without (i–iii) or with (iv–vi) low temperature for 6 h. MYC70pro:GUS expression in mature rosetta leaves (i, iv), base of rosetta leaves (ii,v), and floral organs (iii, vi). ( C ) Subcellular localization of the GFP-MYC proteins. The nuclei were stained with 20 mg/ml PI. The scale bar indicates 20 μm. ( D ) GFP fluorescence at the root tips. Seven-day old seedlings were treated at 4 °C for 3 h, and GFP fluorescence was observed by confocal microscopy. The scale bar indicates 150 μm.

Techniques Used: Expressing, Staining, Fluorescence, Confocal Microscopy

MYC67 and MYC70 interact with ICE1. Yeast 2-hybrid analysis. ( A ) A schematic representation of the bait (top) and prey (bottom) combinations. The yeast strains contained pAS2-ICE1-C (amino acids 267–494) as bait together with pACT-MYC67 m2 (amino acids 3–358), pACT2-MYC67 full (amino acids 1–358), pACT2-MYC70 m4 (amino acids 246–359), or pACT2-MYC70 full (amino acids1–359) as prey. pAS2-1 and pACT2 were used as negative controls. ( B ) The yeast strains were grown on synthetic dropout (SD) plates without leucine and tryptophan (−LW) or on selection plates (SP), which consisted of an SD plate containing X-α-gal and lacking leucine, tryptophan, histidine and adenine. ( C ) The domain structures of MYC67 and MYC70. Acidic, basic, helix-loop-helix (HLH), ZIP domains are presented. Black bars indicate the region of MYC67 m2 (amino acids 3–358) or MYC70 m4 (amino acids 246–359), respectively. ( D ) BiFC analysis indicated interaction between ICE1 and the MYC proteins. ICE1 was fused to N-terminal EYFP (enhanced yellow fluorescent protein), and the MYC proteins were fused to C-terminal EYFP. Each combination of constructs was transfected into Arabidopsis protoplasts. Differential interference contrast (DIC) images and the YFP fluorescence from the BiFC analysis are shown along with the merged image. The scale bar indicates 10 μm.
Figure Legend Snippet: MYC67 and MYC70 interact with ICE1. Yeast 2-hybrid analysis. ( A ) A schematic representation of the bait (top) and prey (bottom) combinations. The yeast strains contained pAS2-ICE1-C (amino acids 267–494) as bait together with pACT-MYC67 m2 (amino acids 3–358), pACT2-MYC67 full (amino acids 1–358), pACT2-MYC70 m4 (amino acids 246–359), or pACT2-MYC70 full (amino acids1–359) as prey. pAS2-1 and pACT2 were used as negative controls. ( B ) The yeast strains were grown on synthetic dropout (SD) plates without leucine and tryptophan (−LW) or on selection plates (SP), which consisted of an SD plate containing X-α-gal and lacking leucine, tryptophan, histidine and adenine. ( C ) The domain structures of MYC67 and MYC70. Acidic, basic, helix-loop-helix (HLH), ZIP domains are presented. Black bars indicate the region of MYC67 m2 (amino acids 3–358) or MYC70 m4 (amino acids 246–359), respectively. ( D ) BiFC analysis indicated interaction between ICE1 and the MYC proteins. ICE1 was fused to N-terminal EYFP (enhanced yellow fluorescent protein), and the MYC proteins were fused to C-terminal EYFP. Each combination of constructs was transfected into Arabidopsis protoplasts. Differential interference contrast (DIC) images and the YFP fluorescence from the BiFC analysis are shown along with the merged image. The scale bar indicates 10 μm.

Techniques Used: Selection, Bimolecular Fluorescence Complementation Assay, Construct, Transfection, Fluorescence

MYC67 and MYC70 were able to bind to the promoter region of CBF3 / DREB1A . ( A ) A schematic representation of the promoter region of CBF3 / DREB1A and the region that was amplified after the ChIP analysis. ( B ) The wild-type and transgenic plants used for the ChIP assay were treated at 4 °C for 3 h. The immunoprecipitated DNA with anti-DYKDDDDK tag monoclonal antibody was quantified by quantitative PCR using primers in the promoter region. The data are the average of technical triplicates for the quantitative PCR (mean ± SD). ( C ) The wild-type and myc mutants were treated at 4 °C for 3 h, and ChIP analysis was performed with anti-ICE1 antibody.
Figure Legend Snippet: MYC67 and MYC70 were able to bind to the promoter region of CBF3 / DREB1A . ( A ) A schematic representation of the promoter region of CBF3 / DREB1A and the region that was amplified after the ChIP analysis. ( B ) The wild-type and transgenic plants used for the ChIP assay were treated at 4 °C for 3 h. The immunoprecipitated DNA with anti-DYKDDDDK tag monoclonal antibody was quantified by quantitative PCR using primers in the promoter region. The data are the average of technical triplicates for the quantitative PCR (mean ± SD). ( C ) The wild-type and myc mutants were treated at 4 °C for 3 h, and ChIP analysis was performed with anti-ICE1 antibody.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Transgenic Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction

40) Product Images from "The Specialized Roles in Carotenogenesis and Apocarotenogenesis of the Phytoene Synthase Gene Family in Saffron"

Article Title: The Specialized Roles in Carotenogenesis and Apocarotenogenesis of the Phytoene Synthase Gene Family in Saffron

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2019.00249

Three dimensional models and location of the saffron phytoene synthase enzymes. (A) Three-dimensional models of the CsPSY enzymes. The models for CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 were created using the PPM Server ( http://opm.phar.umich.edu/server.php ). The α-helices, and loops are depicted as pink and blue, respectively. Blue dots indicate the membrane surface. (B) Subcellular localization of GFP fusion proteins of CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 in agro-infiltrated tobacco leaves after 5 days as detected with confocal laser scanning microscopy and enhanced green fluorescent protein (eGFP) expression. Chlorophyll auto-fluorescence in red (left panel), eGFP fluorescence is shown in green (middle panel) and a merged overlay of the eGFP/chlorophyll fluorescence (right panel) is shown in yellow.
Figure Legend Snippet: Three dimensional models and location of the saffron phytoene synthase enzymes. (A) Three-dimensional models of the CsPSY enzymes. The models for CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 were created using the PPM Server ( http://opm.phar.umich.edu/server.php ). The α-helices, and loops are depicted as pink and blue, respectively. Blue dots indicate the membrane surface. (B) Subcellular localization of GFP fusion proteins of CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 in agro-infiltrated tobacco leaves after 5 days as detected with confocal laser scanning microscopy and enhanced green fluorescent protein (eGFP) expression. Chlorophyll auto-fluorescence in red (left panel), eGFP fluorescence is shown in green (middle panel) and a merged overlay of the eGFP/chlorophyll fluorescence (right panel) is shown in yellow.

Techniques Used: Confocal Laser Scanning Microscopy, Expressing, Fluorescence

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Amplification:

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Synthesized:

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Quantitative RT-PCR:

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Reverse Transcription Polymerase Chain Reaction:

Article Title: Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression
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Real-time Polymerase Chain Reaction:

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Transgenic Assay:

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Luciferase:

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Construct:

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Article Title: The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning
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Polymerase Chain Reaction:

Article Title: RNA-seq profiling reveals differentially expressed genes as potential markers for vital reaction in skin contusion: a pilot study
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Article Title: The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning
Article Snippet: .. DNA constructs and transgenics To generate the transformation vector pUASp-SvbAct::GFP, a fragment without the exon1S and the 5' of the exon2A to the proteolytic cleavage site was amplified by PCR from pUASp-Svb::GFP ( ) and cloned into the pUASp-Svb::GFP, linearized with SpeI and EcoRI, using the In-Fusion HD Cloning kit (Clontech). .. To obtain the pUASp-Svb-3Kmut-GFP, the EcoRI fragment with the 3 K mutated from pAc-SvbK7 ( ) was cloned into the pUASp-Svb::GFP, linearized with EcoRI.

Article Title: A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells
Article Snippet: .. Establishment of transgenic parasite lines To add an hemagglutinin (HA) epitope tag to the endogenous loci of PPM2A (TGGT1_232340), PPM2B (TGGT1_267100), PPM3D (TGGT1_202610), PPM5C(TGGT1_281580), and PPM11 (TGGT1_304955), genomic DNA fragments including the region of the target gene immediately preceding the stop codon were amplified by PCR, and cloned into the p3xHA9-LIC-DHFR vector using the In-Fusion HD Cloning Kit (Takara Bio USA, Inc.). .. The resulting plasmid was linearized within the genomic DNA fragment and transfected into ∆ku80 parasites.

Article Title: Functional interaction between TATA and upstream CACGTG elements regulates the temporally specific expression of Otx mRNAs during early embryogenesis of the sea urchin, Hemicentrotus pulcherrimus
Article Snippet: .. The 800 bp DNA fragment encoding the region from immediately 3′ of the transcriptional initiation site of the HpOtxE promoter to the middle of the open reading frame (ORF) of firefly luciferase was amplified from 0.3 µg of total RNA using the One Step RNA PCR Kit (AMV) (TaKaRa Biomedicals) and the primers TK1682 and TK1686. .. As a reference, the 190 bp DNA fragment encoding the 3′-untranslated region (3′-UTR) of the ubiquitin gene was amplified using the primer pair TK1591 and TK1592.

Transformation Assay:

Article Title: The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning
Article Snippet: .. DNA constructs and transgenics To generate the transformation vector pUASp-SvbAct::GFP, a fragment without the exon1S and the 5' of the exon2A to the proteolytic cleavage site was amplified by PCR from pUASp-Svb::GFP ( ) and cloned into the pUASp-Svb::GFP, linearized with SpeI and EcoRI, using the In-Fusion HD Cloning kit (Clontech). .. To obtain the pUASp-Svb-3Kmut-GFP, the EcoRI fragment with the 3 K mutated from pAc-SvbK7 ( ) was cloned into the pUASp-Svb::GFP, linearized with EcoRI.

Plasmid Preparation:

Article Title: The small 11kDa nonstructural protein of human parvovirus B19 plays a key role in inducing apoptosis during B19 virus infection of primary erythroid progenitor cells
Article Snippet: .. Then we cloned the NS1 ORF (nucleotides [nt's] 616-2631), the 7.5kDa mid-ORF (nt's 2090-2305), and the 11kDa ORF (nt's 4890-5171) into this pGFP plasmid through Eco RI/ Xho I sites as pGFP-NS1, pGFP-7.5kDa, and pGFP-11kDa, respectively. pRFPHA, pRFP-NS1HA, and pRFP-11kDaHA were constructed by replacing GFP with RFPHA (C-terminal HA-tagged red fluorescent protein, DsRed; Clontech), RFP-NS1HA, and RFP-11kDaHA (NS1 and 11kDa were HA tagged at C-terminal) in the pGFP, respectively. .. The 11kDa ORF and the N-terminus encoding sequence (nt's 616-1158) of the NS1 were cloned into pGEX4T3 (GE Healthcare) as pGEX11kDa and pGEXNS1 (amino acids [aa's] 1-181), respectively.

Article Title: The mlpt/Ubr3/Svb module comprises an ancient developmental switch for embryonic patterning
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Article Title: A plasma membrane localized protein phosphatase in Toxoplasma gondii, PPM5C, regulates attachment to host cells
Article Snippet: .. Establishment of transgenic parasite lines To add an hemagglutinin (HA) epitope tag to the endogenous loci of PPM2A (TGGT1_232340), PPM2B (TGGT1_267100), PPM3D (TGGT1_202610), PPM5C(TGGT1_281580), and PPM11 (TGGT1_304955), genomic DNA fragments including the region of the target gene immediately preceding the stop codon were amplified by PCR, and cloned into the p3xHA9-LIC-DHFR vector using the In-Fusion HD Cloning Kit (Takara Bio USA, Inc.). .. The resulting plasmid was linearized within the genomic DNA fragment and transfected into ∆ku80 parasites.

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    TaKaRa in fusion hd cloning kit
    Overview of <t>In-Fusion</t> enzyme-based <t>cloning</t> of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® <t>HD</t> cloning <t>kit</t> with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9
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    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Clone Assay, CRISPR, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Expressing

    Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion ® -based cloning of two gRNA targets for paired nickases (Cas9-D10A). a Illustration of cloning strategy. Schematics of final gRNA cassette is shown in the top panel. Using a set of four universal primers (p1F, p2F, g1R and g2R) and four target-specific primers (g1F and p1R for protospacer target 1, and g2F and p2R for protospacer target 2), four fragments, A, B, C and D are PCR amplified using pEn-Chimera-ccdB plasmid in Round 1 PCR. In Round 2 PCR, using primers p1F and g1R, fragments A and B are fused resulting in fragment AB, and using primers p2F and g2R, fragments C and D are fused resulting in fragment CD. In Step 3, fragments AB and CD are cloned into pDe-Cas9-D10A or pUC57GW using the In-Fusion ® HD cloning system. b A representative gel picture showing PCR fragments of YFP upper panel, SlMLO1, NbPDS and mCherry lower panel. Expected sizes of each fragment are shown on the left. c Protospacer sequences of the targeted genes ( YFP upper panel, NbPDS middle panel, and mCherry lower panel) are highlighted in purple background and the PAM sequences NGG in red background

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

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

    Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Construction and schematic of plasmid. a pEn-Chimera-ccdB. A cassette consisting of chloramphenicol resistance gene ( CmR ) and the ccdB gene was PCR-amplified and inserted between the AtU6-26(P) promoter and the sgRNA of pEn-Chimera [ 22 ] using the In-Fusion ® HD cloning strategy as described in “ Methods ” section. Plasmid pEn-Chimera-ccdB is used as template in PCR for fusing the 20-nucleotide protospacer sequence to the AtU6-26 promoter and sgRNA. Using the ccdB gene virtually eliminated any background colonies, which could arise due to incomplete digestion of pEn-Chimera using the restriction enzymes-based cloning method. b pDe-Cas9-D10A-2 gRNA: Schematic illustration of pDe-Cas9-D10A after two gRNA constructs, gRNA1 and gRNA2, are directly cloned in this vector using the In-Fusion ® HD cloning system. c pUC57GW: this is an in-house constructed Gateway ® -compatible Entry vector, which, in contrast to commonly used Gateway ® Entry/DONR vectors, contains the ccdB and Chloranphenicol ( CmR ) resistance genes. This unique design allows efficient cloning of gRNAs constructs in this vector using the In-Fusion ® HD cloning system without any background colonies. Please see “ Methods ” and “ Results ” section for details

    Article Snippet: Expected fragments (ccdB and CmR genes, 1569 bp and pEN-Chimera backbone, 3738 bp) were gel purified (QIAquick Gel Extraction Kit, Cat#28706, Qiagen) and cloned together using the In-Fusion® HD cloning Kit (Cat#639648, Clontech) according to manufacturer’s instructions.

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing, Construct

    Implementation of TALE–VAS binary expression systems in vivo. a Schematic representation of the TALE–VAS constructs used in flies. The elav 1.8kb , ase 0.8kb , mhc 2.4kb , and repo 1.9kb enhancers were cloned upstream of TALE 1, 3, 4 , and the corresponding 3× VAS sequences were placed in front of Citrine, mCherry, and Cerulean. b – i TALE–VAS-based targeted gene expression in single tissues. Insets show imaging location within each target tissue (red frame). b Dorsal view of elav 1.8kb - TALE 1 > VAS 1 - Citrine-HA third instar larval ventral nerve cord (VNC) stained with anti-HA (transgene expression) and anti-Elav antibody (neuronal marker). c Close up illustrating the overlap between Citrine-HA and nuclear Elav staining in neurons (yellow dashed line and arrow). Some Elav-positive cells lack Citrine-HA expression (yellow asterisk). d Ventral view of ase 0.8kb - TALE 3 > VAS 3 - V5-mCherry third instar larval VNC showing mCherry expression in neuroblasts (red) and Prospero staining of ganglion mother cells (anti-Pros antibody, green). e Detailed, high magnification of a single neuroblast (yellow dashed outline and arrow) and surrounding ganglion mother cells (green). f Third instar larval body wall musculature from mhc 2.4kb - TALE 3 > VAS 3 - V5-mCherry at low magnification. g High-magnification sarcomere morphology showing alternating bands of mCherry expression and F-actin characteristic of striated muscles. h repo 1.9kb - TALE 4 > VAS 4 FLAG-Cerulean third instar larval VNC stained with an anti-GFP antibody (transgene expression) and anti-Repo (glia marker). i Detailed view of subperineural glia showing cells that stain positive for both Cerulean and Repo (yellow dashed outline and arrow) express only Cerulean (blue diamond) or only Repo (yellow asterisk). Scale bars = 50 µm ( b , d , f , h ), 10 µm ( c , e , g , i )

    Journal: Nature Communications

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies

    doi: 10.1038/s41467-017-01592-3

    Figure Lengend Snippet: Implementation of TALE–VAS binary expression systems in vivo. a Schematic representation of the TALE–VAS constructs used in flies. The elav 1.8kb , ase 0.8kb , mhc 2.4kb , and repo 1.9kb enhancers were cloned upstream of TALE 1, 3, 4 , and the corresponding 3× VAS sequences were placed in front of Citrine, mCherry, and Cerulean. b – i TALE–VAS-based targeted gene expression in single tissues. Insets show imaging location within each target tissue (red frame). b Dorsal view of elav 1.8kb - TALE 1 > VAS 1 - Citrine-HA third instar larval ventral nerve cord (VNC) stained with anti-HA (transgene expression) and anti-Elav antibody (neuronal marker). c Close up illustrating the overlap between Citrine-HA and nuclear Elav staining in neurons (yellow dashed line and arrow). Some Elav-positive cells lack Citrine-HA expression (yellow asterisk). d Ventral view of ase 0.8kb - TALE 3 > VAS 3 - V5-mCherry third instar larval VNC showing mCherry expression in neuroblasts (red) and Prospero staining of ganglion mother cells (anti-Pros antibody, green). e Detailed, high magnification of a single neuroblast (yellow dashed outline and arrow) and surrounding ganglion mother cells (green). f Third instar larval body wall musculature from mhc 2.4kb - TALE 3 > VAS 3 - V5-mCherry at low magnification. g High-magnification sarcomere morphology showing alternating bands of mCherry expression and F-actin characteristic of striated muscles. h repo 1.9kb - TALE 4 > VAS 4 FLAG-Cerulean third instar larval VNC stained with an anti-GFP antibody (transgene expression) and anti-Repo (glia marker). i Detailed view of subperineural glia showing cells that stain positive for both Cerulean and Repo (yellow dashed outline and arrow) express only Cerulean (blue diamond) or only Repo (yellow asterisk). Scale bars = 50 µm ( b , d , f , h ), 10 µm ( c , e , g , i )

    Article Snippet: The Citrine responder (pJFRC81_3×VAS1 -Syn21-Citrine-HA-P10) was assembled from plasmid pJFRC81_3×VAS-1-GFP-P10 digested with BglII and EcoRI, and fragments Syn21-Citrine-HA, VAS-1-HA-P10, Mhc-SD by in-fusion cloning (Clontech, 639648).

    Techniques: Expressing, In Vivo, Construct, Clone Assay, Imaging, Staining, Marker

    High-specificity multiplex transgene expression in Drosophila nervous system. a Diagrammatic representation of the experimental framework used for TALE–VAS-mediated parallel transgene expression in multiple tissues in vivo. Tissue-specific expression of three independent transgenes is achieved in the F0 generation by crossing combinatorial driver and responder lines. b Simultaneous expression of mCherry in neuroblasts (red), Citrine in neurons (green), and Cerulean in surface glia cells (white) by three independent TALE–VAS pairs in a single Drosophila larval CNS ( elav 1.8kb -TALE 1 , ase 0.8kb -TALE 3 , repo 1.9kb -TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean ; dorsal view of unfixed CNS). c High-magnification imaging of the right optic lobe from the same genotype in b illustrating the three distinct populations of labelled neighbouring cells (neuroblasts, neurons, and glial cells). d High-magnification imaging of a larval segmental nerve showing an axonal bundle (Citrine, green) ensheathed by a glial cell (Cerulean, white). The ase 0.8kb -TALE 3 driver is inactive in this tissue. e The NMJ innervating muscle 6 and 7 from elav 1.8kb -TALE 1 , mhc 2.4kb -TALE 3 , repo 1.9kb - TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean larvae. The post-synaptic muscle field is labelled by mCherry expression (red) and the neuronal pre-synaptic terminal by Citrine (green). The repo 1.9kb - TALE 4 driver is inactive in this tissue. Scale bars = 100 µm ( b ), 25 µm ( c , e ), 10 µm ( d )

    Journal: Nature Communications

    Article Title: A multiplexable TALE-based binary expression system for in vivo cellular interaction studies

    doi: 10.1038/s41467-017-01592-3

    Figure Lengend Snippet: High-specificity multiplex transgene expression in Drosophila nervous system. a Diagrammatic representation of the experimental framework used for TALE–VAS-mediated parallel transgene expression in multiple tissues in vivo. Tissue-specific expression of three independent transgenes is achieved in the F0 generation by crossing combinatorial driver and responder lines. b Simultaneous expression of mCherry in neuroblasts (red), Citrine in neurons (green), and Cerulean in surface glia cells (white) by three independent TALE–VAS pairs in a single Drosophila larval CNS ( elav 1.8kb -TALE 1 , ase 0.8kb -TALE 3 , repo 1.9kb -TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean ; dorsal view of unfixed CNS). c High-magnification imaging of the right optic lobe from the same genotype in b illustrating the three distinct populations of labelled neighbouring cells (neuroblasts, neurons, and glial cells). d High-magnification imaging of a larval segmental nerve showing an axonal bundle (Citrine, green) ensheathed by a glial cell (Cerulean, white). The ase 0.8kb -TALE 3 driver is inactive in this tissue. e The NMJ innervating muscle 6 and 7 from elav 1.8kb -TALE 1 , mhc 2.4kb -TALE 3 , repo 1.9kb - TALE 4 > VAS 1 -Citrine-HA , VAS 3 -V5-mCherry , VAS 4 -FLAG-Cerulean larvae. The post-synaptic muscle field is labelled by mCherry expression (red) and the neuronal pre-synaptic terminal by Citrine (green). The repo 1.9kb - TALE 4 driver is inactive in this tissue. Scale bars = 100 µm ( b ), 25 µm ( c , e ), 10 µm ( d )

    Article Snippet: The Citrine responder (pJFRC81_3×VAS1 -Syn21-Citrine-HA-P10) was assembled from plasmid pJFRC81_3×VAS-1-GFP-P10 digested with BglII and EcoRI, and fragments Syn21-Citrine-HA, VAS-1-HA-P10, Mhc-SD by in-fusion cloning (Clontech, 639648).

    Techniques: Multiplex Assay, Expressing, In Vivo, Imaging

    Standard assembly versus In-Fusion assembly. ( a ) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. ( b ) In-Fusion assembly of two BioBricks involves PCR, purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick prefix or suffix homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format.

    Journal: Nucleic Acids Research

    Article Title: In-Fusion BioBrick assembly and re-engineering

    doi: 10.1093/nar/gkq179

    Figure Lengend Snippet: Standard assembly versus In-Fusion assembly. ( a ) Standard Assembly of two BioBricks (Parts A and B) involves restriction digestion and ligation. Both parts are on pSB1A2 vectors encoding ampicillin resistance. The Part A plasmid is digested with EcoRI (E) and SpeI (S), while the second plasmid is digested with EcoRI (E) and XbaI (X). SpeI and XbaI restricted fragments have compatible sticky ends for ligation. The desired digested fragments are gel purified and ligated together to create the assembled plasmid with both parts. A scar sequence is left between both parts that does not have the original restriction site and the restriction sites flanking the parts are maintained. ( b ) In-Fusion assembly of two BioBricks involves PCR, purification, and a subsequent In-Fusion reaction. Parts A and B are PCR-amplified (in this example the vector is amplified with Part B) and purified without gel extraction. Each assembly requires four primers, where two are specific to the junction (parts to assemble) and two are general vector primers. BioBrick Part A (blue) and Part B (red) are on pSB1A2 plasmids encoding ampicillin resistance. Primers described in ‘Materials and Methods’ section are color-coded to show their homology. The thick black line indicates BioBrick prefix or suffix homology on the pSB1A2 vector. The yellow sequence is the scar that is normally between parts after standard BioBrick assembly, if this is desired, or can be a linker sequence for a fusion protein. The purified PCR products are fused together in the In-Fusion reaction to create a circular plasmid. Restriction sites flanking the parts maintain the standard BioBrick format.

    Article Snippet: The method described here is an alternative assembly strategy that allows for two or more PCR-amplified BioBricks to be quickly assembled and re-engineered using the Clontech In-Fusion PCR Cloning Kit.

    Techniques: Ligation, Plasmid Preparation, Purification, Sequencing, Polymerase Chain Reaction, Amplification, Gel Extraction