apali  (New England Biolabs)


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    Name:
    ApaLI
    Description:
    ApaLI 12 500 units
    Catalog Number:
    r0507l
    Price:
    269
    Size:
    12 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs apali
    ApaLI
    ApaLI 12 500 units
    https://www.bioz.com/result/apali/product/New England Biolabs
    Average 93 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    apali - by Bioz Stars, 2020-07
    93/100 stars

    Images

    1) Product Images from "Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein"

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.780239

    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.
    Figure Legend Snippet: Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Techniques Used: Footprinting, Construct, Plasmid Preparation, Binding Assay, Sequencing

    2) Product Images from "Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease"

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp643

    Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).
    Figure Legend Snippet: Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).

    Techniques Used: Southern Blot, Clone Assay, Transfection, Derivative Assay, Plasmid Preparation, Molecular Weight, Amplification, Binding Assay, Marker

    PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.
    Figure Legend Snippet: PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.

    Techniques Used: Polymerase Chain Reaction, Genetically Modified, Stable Transfection, Nested PCR, Clone Assay, Derivative Assay, Transfection, Plasmid Preparation, Labeling, Amplification, Negative Control, Marker, Molecular Weight, Agarose Gel Electrophoresis

    3) Product Images from "Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells"

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2014.2804

    Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.
    Figure Legend Snippet: Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.

    Techniques Used: Activation Assay, Western Blot, Incubation, Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    4) Product Images from "RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation"

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp780

    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.
    Figure Legend Snippet: Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Techniques Used: Southern Blot, Staining, Produced, Generated

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Different allelic frequency of progressive rod-cone degeneration in two populations of Labrador Retrievers in Japan
    Article Snippet: .. The PCR product was digested with 5 units of ApaLI (New England Biolab, MA, U.S.A.) at 37°C for 90 min to yield 151 and 81-bp fragments in the wild genotype (GG), but not in the mutant genotype (AA). .. After digestion, the PCR products were electrophoresed on a 2% agarose gel and visualized by UltraPowerTM DNA Dye solution (Gelex International, Tokyo, Japan).

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells
    Article Snippet: .. ER stress was assessed by detecting activated XBP-1 , which was analyzed by digestion of PCR products with ApaLI (New England Biolabs Inc., MA, USA), as previously described ( ). .. Because the ApaLI site in XBP-1 mRNA is spliced out upon activation, activated XBP-1 shows one large band after ApaLI digestion, while inactivated XBP-1 shows two ApaLI-digested bands.

    Mutagenesis:

    Article Title: Different allelic frequency of progressive rod-cone degeneration in two populations of Labrador Retrievers in Japan
    Article Snippet: .. The PCR product was digested with 5 units of ApaLI (New England Biolab, MA, U.S.A.) at 37°C for 90 min to yield 151 and 81-bp fragments in the wild genotype (GG), but not in the mutant genotype (AA). .. After digestion, the PCR products were electrophoresed on a 2% agarose gel and visualized by UltraPowerTM DNA Dye solution (Gelex International, Tokyo, Japan).

    Agarose Gel Electrophoresis:

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease
    Article Snippet: .. After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer. .. Next, the DNA was transferred by capillary action to an Amersham Hybond-XL membrane (GE Healthcare) using a standard Southern blot technique.

    Plasmid Preparation:

    Article Title: High-Affinity IgG Antibodies Develop Naturally in Ig-Knockout Rats Carrying Germline Human IgH/Ig\u03ba/Ig\u03bb Loci Bearing the Rat CH Region
    Article Snippet: .. From this plasmid, an ApaLI-BamHI fragment containing URA3 followed by CEN4 or a PmlI–SphI fragment containing CEN4 followed by HygR was cut out and ligated to ApaLI and BamHI or HpaI and SphI doubly digested pBACBelo11 (New England BioLabs) to yield pBelo-CEN-URA and pBelo-CEN-HYG. .. To construct BAC6-VH 3-11, initially two fragments, a 115-kb NotI-PmeI and a 110-kb RsrII-SgrAI, were cut out from the BAC clone 3054M17 CITB.

    Isolation:

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation
    Article Snippet: .. Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min. .. The purified genomic DNA was re-digested with 100 U of ApaI completely (for first digestion with XhoI, PstI and SmaI) or 100 U of PstI (for first digestion with SpeI and ApaLI).

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    New England Biolabs apali
    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with <t>BamHI-HindIII</t> ( A ), BamHI-EcoRI ( B ), and <t>ApaLI</t> ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.
    Apali, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/apali/product/New England Biolabs
    Average 93 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    apali - by Bioz Stars, 2020-07
    93/100 stars
      Buy from Supplier

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    Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Journal: The Journal of Biological Chemistry

    Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein

    doi: 10.1074/jbc.M117.780239

    Figure Lengend Snippet: Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.

    Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.

    Techniques: Footprinting, Construct, Plasmid Preparation, Binding Assay, Sequencing

    Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).

    Journal: Nucleic Acids Research

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    doi: 10.1093/nar/gkp643

    Figure Lengend Snippet: Investigation of targeted versus random chromosomal insertion of exogenous DNA through restriction mapping and Southern blot analyses. ( A ) Diagram of the Southern blot assay performed on ApaLI-digested chromosomal DNA extracted from randomly selected clones of pA1.GFP.A2-transfected HeLa cells. The ApaLI restriction map of the AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. Non-targeted and targeted human chromosome 19 alleles are expected to give rise to 7.1- and 9.9-kb ApaLI DNA fragments, respectively, using the AAVS1 -specific probe (orange horizontal bar). The 9.9-kb DNA species should also be specifically recognized by the transgene-specific probe, which spans the entire hrGFP ORF (green horizontal bar). Both DNA probes are drawn in relation to their target sequences. For an explanation of the other symbols see the legend of Figure 1 . ( B ) Southern blots of ApaLI-digested chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The open arrowheads point at the 9.9-kb DNA fragments expected to emerge following homology-directed gene targeting at AAVS1 . The open arrows mark higher molecular weight DNA species that are also recognized by both probes indicating insertion of exogenous DNA at AAVS1 loci that underwent local Rep78/68-induced DNA amplification/rearrangement. The 12.7-kb ApaLI-linearized pA1.GFP.A2 DNA (solid arrowhead in lower panel) served as an internal control for probe binding specificity and removal. The solid arrowhead in the upper panel points at the 7.1-kb ApaLI fragments derived from unmodified AAVS1 alleles. Marker, Gene Ruler DNA Ladder Mix molecular weight ladder (Fermentas).

    Article Snippet: After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer.

    Techniques: Southern Blot, Clone Assay, Transfection, Derivative Assay, Plasmid Preparation, Molecular Weight, Amplification, Binding Assay, Marker

    PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.

    Journal: Nucleic Acids Research

    Article Title: Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease

    doi: 10.1093/nar/gkp643

    Figure Lengend Snippet: PCR assays to identify cells genetically modified through homology-directed gene targeting. ( A ) Overview of the PCR assays carried out on genomic DNA of stably transduced HeLa cells clonally expanded from cultures exposed (+Rep) or not exposed (−Rep) to pGAPDH.Rep78/68. The AAVS1 locus is depicted before and after HR with the donor template pA1.GFP.A2. HR-mediated transgene insertion should yield 2.7-kb PCR products using primers #649 and #651 (horizontal dark blue bar). The specificity of these amplicons can be confirmed by semi-nested PCR using primers #649 and #186 combined with restriction fragment length analyses of the resulting 2.3-kb DNA species (horizontal brown bars). The numbers above the brown bars refer to restriction fragment sizes. Open circle, prokaryotic origin of replication. For an explanation of the other symbols see the legend of Figure 1 . ( B ) PCR analysis carried out on chromosomal DNA of parental HeLa cells (HeLa) and of clones derived from HeLa cells co-transfected with pA1.GFP.A1 and pGAPDH.Rep78/68 (+Rep) or with pA1.GFP.A2 and ‘empty plasmid’ (−Rep). The panels labeled GT display the results of amplification reactions performed with primers #649 and #651, which are specific for the AAVS1 locus and the EF1α promoter, respectively. PCR amplifications targeting a 1.9-kb segment of the HPRT1 were carried out in parallel to ascertain the integrity of the genomic DNA corresponding to individual HeLa cell clones (see panels marked HPRT1). PCR mixtures containing water instead of DNA served as a negative control (H 2 O). The positions and sizes (in kilo base pairs) of specific PCR products are indicated at the right. Marker, Gene Ruler DNA Ladder Mix molecular weight marker (Fermentas). ( C ) Characterization of the 2.7-kb amplification products obtained with primers #649 and #651 by semi-nested PCR and restriction enzyme fragment length analyses. A semi-nested PCR using primers #649 and #186 was performed on the 2.7-kb amplicons corresponding to clones 2, 4 and 5. The resulting 2.3-kb PCR products were subjected to agarose gel electrophoresis after digestion with ApaLI, PauI or SmaI. Und., undigested PCR fragments.

    Article Snippet: After overnight digestion with ApaLI (New England Biolabs), 10 µg DNA samples were resolved in a 0.8% agarose gel in 1× Tris–acetate–EDTA buffer.

    Techniques: Polymerase Chain Reaction, Genetically Modified, Stable Transfection, Nested PCR, Clone Assay, Derivative Assay, Transfection, Plasmid Preparation, Labeling, Amplification, Negative Control, Marker, Molecular Weight, Agarose Gel Electrophoresis

    Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.

    Journal: International Journal of Oncology

    Article Title: Shikonin, dually functions as a proteasome inhibitor and a necroptosis inducer in multiple myeloma cells

    doi: 10.3892/ijo.2014.2804

    Figure Lengend Snippet: Accumulation of ubiquitinated proteins and activation of XBP-1 by SHK. (A) Left panel, western blot analyses of ubiquitinated proteins. U266 and KMS-12-PE cells were incubated with 2.5 or 5 μM SHK for 7 h. SHK induced an accumulation of ubiquitinated proteins in a dose-dependent manner. Right panel, inhibition of 20S chymotrypsin-like activity by SHK. SHK decreased 20S chymotrypsin-like activity at a dose-dependent manner. (B) Activation of XBP-1 by SHK. XBP-1 mRNA from U266 and KMS-12-PE cells treated with SHK was converted to cDNA by RT-PCR and then subsequently digested with ApaLI to distinguish inactive and active XBP-1 . Thapsigargin (Thap) was used as an endoplasmic reticulum stress inducer at 100 nM for 8 h. SHK was used at concentrations of 2.5–5 μM for 2 h. The longer fragment derived from the active form of XBP-1 mRNA (upper band) and two shorter bands derived from the inactive form (middle and lower bands) were detected. A decrease of the inactive bands was found in U266 (left panel) cells, while increase of active band was noted in KMS-12-PE cells (right panel) by treatment with SHK. (C) Relative density of active XBP-1 compared with inactive XBP-1, as shown in (B) was calculated. A significant increase of active XBP-1 was found by treatment with SHK in U266 cells. KMS-12-PE cells showed a similar trend, although the difference was not statistically significant.

    Article Snippet: ER stress was assessed by detecting activated XBP-1 , which was analyzed by digestion of PCR products with ApaLI (New England Biolabs Inc., MA, USA), as previously described ( ).

    Techniques: Activation Assay, Western Blot, Incubation, Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Journal: Nucleic Acids Research

    Article Title: RIP140 in thyroid hormone-repression and chromatin remodeling of Crabp1 gene during adipocyte differentiation

    doi: 10.1093/nar/gkp780

    Figure Lengend Snippet: Alterations of restriction enzyme accessibility in the juxtaposed region of Crabp1 promoter along the course of adipocyte differentiation. ( A ) Detection of nucleosome array. Chromatin of MNase partially digested nuclei was separated on agarose gels followed by Southern blot analysis (left panel) and EtBr staining (right panel). A probe used covering the TIS (HinfI-PvuII) was used for Southern blot analysis. ( B–F ) Nuclei (differentiation Days 0, 4, 8) were digested with the indicated restriction enzymes and the recovered chromatin DNAs were completely re-digested with ApaI ( B–D ) or PstI ( E and F ) (the relationship of specific restriction site and each individual nucleosome was depicted in panel G), followed by Southern blot analyses using probe 1 ( D ) or probe 2 (B–C and E–F). ( G ) Schematic description of restriction enzyme digestion, nucleosomes, and predicted fragments detected with probes on the Southern blots. Complete digestion with ApaI produced 1.3 kb, and complete digestion with PstI produced 1.65 kb fragments. The generated fragments by digestion with XhoI, PstI, SmaI, SpeI and ApaLI for the corresponding nucleosome are 0.93, 0.67, 0.86, 0.55 and 0.65 kb, respectively. Nucleosomes on this array were named N5 to N-1, from the 5′- to the 3′-ends of this chromatin segment. Experiments were performed for least three times.

    Article Snippet: Isolated nuclei from differentiating 3T3-L1 cells were digested with 100 U of PstI, XhoI, SmaI, SpeI and ApaLI (New England Biolabs) for 30 min.

    Techniques: Southern Blot, Staining, Produced, Generated