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    Millipore dna binding
    Annealing-promoted religation of the cleaved C25-Dabcyl-Cy3 <t>DNA</t> by <t>Sso</t> topo III at various temperatures. Sso topo III (2 μM) was incubated with C25-Dabcyl-Cy3 (40 nM) for 40 min at 25 °C in the presence of MgCl 2 (1 mM). EDTA (5 mM) was added to chelate Mg 2+ . The cleavage intermediates were allowed to cool down from 75 °C to indicated temperatures at 0.3 °C/s in the presence of EDTA (5 mM) and the complementary strand NC25 at an NC25/C25-Dabcyl-Cy3 molar ratio of 3. Religation of the cleaved strand was initiated by the addition of MgCl 2 (30 mM) at 37, 40, 45, 50 or 55 °C, and the change in fluorescence was measured in a stopped-flow spectrometer. The data were fitted to first-order exponential function. Each curve is derived from 5~6 independent measurements.
    Dna Binding, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna binding properties
    Differential binding to the hsp70 heat shock element (HSE) maps to the heat shock transcription factor (HSF) <t>DNA-binding</t> domain. ( A ) DNase I footprinting by mHSF1 and mHSF2 to the hsp70 HSE. The hsp70 HSE probe was labeled at the 5′ end of the coding strand, and the concentration of the probe in all reaction mixtures was 0.1 nM. The amounts of bacterial lysates in lanes A–E were 0.5, 1, 2, 4, and 8 μL, respectively. Control reaction shows DNase I footprinting reaction in the absence of protein. The extent of <t>HSF1</t> and HSF2 protection are indicated at the left with brackets. The positions of HSEs 1 to 5 are marked by arrows. ( B ) Specificity in DNA-binding maps to the DNA-binding domain. Chimeras in which the DBD was interchanged were expressed and purified as in Materials and Methods and used for DNase I footprinting. Lane assignments are as described for panel A . The extent of protein binding is delimited by the brackets.
    Dna Binding Properties, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna binding dye 4
    Differential binding to the hsp70 heat shock element (HSE) maps to the heat shock transcription factor (HSF) <t>DNA-binding</t> domain. ( A ) DNase I footprinting by mHSF1 and mHSF2 to the hsp70 HSE. The hsp70 HSE probe was labeled at the 5′ end of the coding strand, and the concentration of the probe in all reaction mixtures was 0.1 nM. The amounts of bacterial lysates in lanes A–E were 0.5, 1, 2, 4, and 8 μL, respectively. Control reaction shows DNase I footprinting reaction in the absence of protein. The extent of <t>HSF1</t> and HSF2 protection are indicated at the left with brackets. The positions of HSEs 1 to 5 are marked by arrows. ( B ) Specificity in DNA-binding maps to the DNA-binding domain. Chimeras in which the DBD was interchanged were expressed and purified as in Materials and Methods and used for DNase I footprinting. Lane assignments are as described for panel A . The extent of protein binding is delimited by the brackets.
    Dna Binding Dye 4, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Annealing-promoted religation of the cleaved C25-Dabcyl-Cy3 DNA by Sso topo III at various temperatures. Sso topo III (2 μM) was incubated with C25-Dabcyl-Cy3 (40 nM) for 40 min at 25 °C in the presence of MgCl 2 (1 mM). EDTA (5 mM) was added to chelate Mg 2+ . The cleavage intermediates were allowed to cool down from 75 °C to indicated temperatures at 0.3 °C/s in the presence of EDTA (5 mM) and the complementary strand NC25 at an NC25/C25-Dabcyl-Cy3 molar ratio of 3. Religation of the cleaved strand was initiated by the addition of MgCl 2 (30 mM) at 37, 40, 45, 50 or 55 °C, and the change in fluorescence was measured in a stopped-flow spectrometer. The data were fitted to first-order exponential function. Each curve is derived from 5~6 independent measurements.

    Journal: Scientific Reports

    Article Title: Kinetic insights into the temperature dependence of DNA strand cleavage and religation by topoisomerase III from the hyperthermophile Sulfolobus solfataricus

    doi: 10.1038/s41598-017-05837-5

    Figure Lengend Snippet: Annealing-promoted religation of the cleaved C25-Dabcyl-Cy3 DNA by Sso topo III at various temperatures. Sso topo III (2 μM) was incubated with C25-Dabcyl-Cy3 (40 nM) for 40 min at 25 °C in the presence of MgCl 2 (1 mM). EDTA (5 mM) was added to chelate Mg 2+ . The cleavage intermediates were allowed to cool down from 75 °C to indicated temperatures at 0.3 °C/s in the presence of EDTA (5 mM) and the complementary strand NC25 at an NC25/C25-Dabcyl-Cy3 molar ratio of 3. Religation of the cleaved strand was initiated by the addition of MgCl 2 (30 mM) at 37, 40, 45, 50 or 55 °C, and the change in fluorescence was measured in a stopped-flow spectrometer. The data were fitted to first-order exponential function. Each curve is derived from 5~6 independent measurements.

    Article Snippet: The kinetics of DNA binding by Sso topo III was determined by rapidly mixing equal volumes (50 μl) of Cy3-Dabcyl-labeled C25 DNA (8 nM) and Y318F (320 nM) in buffer A 50 mM Tris-HCl, pH 8.8, 90 mM NaCl, 0.1 mM EDTA, pH 8.0, 2.5 mM MgCl2 and 0.01% Tween 20 (Sigma, Germany) on the stopped-flow apparatus at an indicated temperature.

    Techniques: Incubation, Fluorescence, Flow Cytometry, Derivative Assay

    (A) Morphological changes of stigmasterol treated HepG2 cells. For observation of morphological changes, cultured HepG2 cells were treated with stigmasterol for 24 h and morphological changes were detected under a light microscope (viewed at magnification of 100×). (B) Fluorescence micrographs showing the stigmasterol induced DNA damage. Stigmasterol treated cells were stained with Hoechst 33342 dye and detected under fluorescence microscope (viewed at magnification of 400×). The blue fluorescence in the nucleus indicates DNA fragmentation. (C) Cell numbers were counted using FACS after Hoechst 33342 staining. (Red; blank, Blue; 5 μM, Yellow; 10 μM, Green; 20 μM) (D) Flow cytometric analysis of the effect of stigmasterol in HepG2 cells experimented using Annexin V-PI staining assay.

    Journal: BMB Reports

    Article Title: Stigmasterol isolated from marine microalgae Navicula incerta induces apoptosis in human hepatoma HepG2 cells

    doi: 10.5483/BMBRep.2014.47.8.153

    Figure Lengend Snippet: (A) Morphological changes of stigmasterol treated HepG2 cells. For observation of morphological changes, cultured HepG2 cells were treated with stigmasterol for 24 h and morphological changes were detected under a light microscope (viewed at magnification of 100×). (B) Fluorescence micrographs showing the stigmasterol induced DNA damage. Stigmasterol treated cells were stained with Hoechst 33342 dye and detected under fluorescence microscope (viewed at magnification of 400×). The blue fluorescence in the nucleus indicates DNA fragmentation. (C) Cell numbers were counted using FACS after Hoechst 33342 staining. (Red; blank, Blue; 5 μM, Yellow; 10 μM, Green; 20 μM) (D) Flow cytometric analysis of the effect of stigmasterol in HepG2 cells experimented using Annexin V-PI staining assay.

    Article Snippet: After observation, the cells, they were stained with 1 μg/ml of the fluorescent DNA-binding dye, Bisbenzimide Hoechst 33342 (Sigma) and incubated for 1 h to reveal nuclear condensation/aggregation.

    Techniques: Cell Culture, Light Microscopy, Fluorescence, Staining, Microscopy, FACS, Flow Cytometry

    Binding of RBP-Jκ and RBP-Jκ/RTA complexes to an RBP-Jκ recognition site. The indicated proteins were cloned into a T7 transcription vector and expressed in a rabbit reticulocyte lysate (RRL). The RRL extracts programmed with the indicated vectors were added to a 32 P-labeled oligonucleotide corresponding to a canonical RBP-Jκ site, and complexes were analyzed by EMSA. Arrow indicates the position of RBP-Jκ/DNA complex. “Vector” refers to lysate programmed with an empty T7 transcription vector. WT, wild-type RBP-Jκ site; MT, mutant RBP-Jκ site.

    Journal: Genes & Development

    Article Title: The lytic switch protein of KSHV activates gene expression via functional interaction with RBP-J? (CSL), the target of the Notch signaling pathway

    doi: 10.1101/gad.996502

    Figure Lengend Snippet: Binding of RBP-Jκ and RBP-Jκ/RTA complexes to an RBP-Jκ recognition site. The indicated proteins were cloned into a T7 transcription vector and expressed in a rabbit reticulocyte lysate (RRL). The RRL extracts programmed with the indicated vectors were added to a 32 P-labeled oligonucleotide corresponding to a canonical RBP-Jκ site, and complexes were analyzed by EMSA. Arrow indicates the position of RBP-Jκ/DNA complex. “Vector” refers to lysate programmed with an empty T7 transcription vector. WT, wild-type RBP-Jκ site; MT, mutant RBP-Jκ site.

    Article Snippet: Briefly, proteins from RRL were mixed with 1 μg poly (dI-dC) (Sigma) in 1× DNA binding buffer for 30 min on ice.

    Techniques: Binding Assay, Clone Assay, Plasmid Preparation, Labeling, Mutagenesis

    Condensins I and II display different quantitative dynamics during mitosis as determined by automated FCS-calibrated confocal 3D time-lapse imaging. (A) Genome-edited HK cells with homozygously mEGFP-tagged Condensin subunits and chromosomes stained by SiR-DNA were imaged every 90 s for a total of 60 min by 3D confocal microscopy, which was automatically triggered after prophase onset. Images were calibrated by FCS to convert fluorescence intensities ( FI ) into cellular protein concentration maps ( C ) and cellular protein number maps ( N ; for details, see Fig. S1, D–I). For SMC4 (Condensin I/II), fluorescence intensities, cellular protein concentration, and protein number maps are shown. For CAP-D2 and CAP-H (Condensin I) and CAP-D3 and CAP-H2 (Condensin II), protein number maps are depicted. Single z planes are shown for specific mitotic phases comparable between subunits. Condensin II is nuclear/on chromosomes throughout mitosis. Condensin I is cytoplasmic in prophase and gains access to mitotic chromosomes after NEBD in prometaphase. A Gaussian blur (σ = 1) was applied to the images for presentation purposes. Bar, 10 µm. (B) SMC4 protein numbers in specific compartments of mitotic HK cells (cytoplasm [blue-green], nucleus [orange], chromatin bound [beige], and chromatin soluble [turquoise]) are plotted against the mitotic standard time, and the corresponding mitotic phases are indicated. Shown is the mean of 17 cells from four independent experiments. Cellular landmarks were used for 3D segmentation and conversion of compartment-specific protein concentrations into protein numbers (for details, see Fig. S1 I and Materials and methods). Chromosome-bound and soluble chromatin proteins defined as the freely diffusing proteins within the chromatin volume were distinguished until anaphase as described in Materials and methods. For late mitotic stages, the nuclear compartment could not be divided into chromatin-bound and soluble proteins. (C) The numbers of chromosome-bound (prophase to anaphase) and nuclear Condensin subunits (telophase and cytokinesis) are plotted against the mitotic standard time: Condensin II subunits (CAP-D3, dark green; CAP-H2, light green), Condensin I subunits (CAP-D2, dark magenta; CAP-H, light magenta), and shared SMC4 (dark gray) are shown. Means (colored lines) and SD (light gray areas) are shown. (D) The numbers of chromosome-bound Condensin subunits from C are plotted for selected mitotic stages according to the images shown in A. SMC4 subunits (gray) as well as the sum of corresponding HEAT-repeat subunits (CAP-D2, dark magenta, Condensin I; CAP-D3, dark green, Condensin II) and corresponding kleisin subunits (CAP-H, light magenta, Condensin I; CAP-H2, light green, Condensin II) are shown. For C and D, the means of ∼20 cells per subunit (range: 10–36) from three to seven independent experiments are plotted; 36, 22, 10, 17, and 17 cells from seven, three, five, three, and four experiments for CAP-H, CAP-H2, CAP-D2, CAP-D3, and SMC4, respectively, are shown.

    Journal: The Journal of Cell Biology

    Article Title: A quantitative map of human Condensins provides new insights into mitotic chromosome architecture

    doi: 10.1083/jcb.201801048

    Figure Lengend Snippet: Condensins I and II display different quantitative dynamics during mitosis as determined by automated FCS-calibrated confocal 3D time-lapse imaging. (A) Genome-edited HK cells with homozygously mEGFP-tagged Condensin subunits and chromosomes stained by SiR-DNA were imaged every 90 s for a total of 60 min by 3D confocal microscopy, which was automatically triggered after prophase onset. Images were calibrated by FCS to convert fluorescence intensities ( FI ) into cellular protein concentration maps ( C ) and cellular protein number maps ( N ; for details, see Fig. S1, D–I). For SMC4 (Condensin I/II), fluorescence intensities, cellular protein concentration, and protein number maps are shown. For CAP-D2 and CAP-H (Condensin I) and CAP-D3 and CAP-H2 (Condensin II), protein number maps are depicted. Single z planes are shown for specific mitotic phases comparable between subunits. Condensin II is nuclear/on chromosomes throughout mitosis. Condensin I is cytoplasmic in prophase and gains access to mitotic chromosomes after NEBD in prometaphase. A Gaussian blur (σ = 1) was applied to the images for presentation purposes. Bar, 10 µm. (B) SMC4 protein numbers in specific compartments of mitotic HK cells (cytoplasm [blue-green], nucleus [orange], chromatin bound [beige], and chromatin soluble [turquoise]) are plotted against the mitotic standard time, and the corresponding mitotic phases are indicated. Shown is the mean of 17 cells from four independent experiments. Cellular landmarks were used for 3D segmentation and conversion of compartment-specific protein concentrations into protein numbers (for details, see Fig. S1 I and Materials and methods). Chromosome-bound and soluble chromatin proteins defined as the freely diffusing proteins within the chromatin volume were distinguished until anaphase as described in Materials and methods. For late mitotic stages, the nuclear compartment could not be divided into chromatin-bound and soluble proteins. (C) The numbers of chromosome-bound (prophase to anaphase) and nuclear Condensin subunits (telophase and cytokinesis) are plotted against the mitotic standard time: Condensin II subunits (CAP-D3, dark green; CAP-H2, light green), Condensin I subunits (CAP-D2, dark magenta; CAP-H, light magenta), and shared SMC4 (dark gray) are shown. Means (colored lines) and SD (light gray areas) are shown. (D) The numbers of chromosome-bound Condensin subunits from C are plotted for selected mitotic stages according to the images shown in A. SMC4 subunits (gray) as well as the sum of corresponding HEAT-repeat subunits (CAP-D2, dark magenta, Condensin I; CAP-D3, dark green, Condensin II) and corresponding kleisin subunits (CAP-H, light magenta, Condensin I; CAP-H2, light green, Condensin II) are shown. For C and D, the means of ∼20 cells per subunit (range: 10–36) from three to seven independent experiments are plotted; 36, 22, 10, 17, and 17 cells from seven, three, five, three, and four experiments for CAP-H, CAP-H2, CAP-D2, CAP-D3, and SMC4, respectively, are shown.

    Article Snippet: For tagging SMC4 at the C terminus and CAP-H at the N terminus with mEGFP, zinc finger nucleases (ZFNs) containing DNA binding sequences listed in Table S1 were purchased from Sigma-Aldrich.

    Techniques: Imaging, Staining, Confocal Microscopy, Fluorescence, Protein Concentration

    STED superresolution imaging of Condensins I and II. Condensin subunits were immunostained (anti-GFP), and mitotic cells with chromatids oriented in parallel to the focal plane were selected for imaging. DNA (Hoechst) and Condensins (mEGFP tag and anti-GFP immunostaining) were imaged by diffraction-limited microscopy, whereas immunostained Condensin was also imaged by STED microscopy. Representative images of mitotic chromatids with SMC4 (Condensin I + II), CAP-H (Condensin I), and CAP-H2 (Condensin II) in late prometaphase (left) and early anaphase (right) are shown. For SMC4 (top), whole-cell overview images as well as overlays of superresolved Condensin (white) and diffraction-limited DNA (magenta) imaging are shown. Overlays represent zooms into single chromatids (turquoise box). For CAP-H (middle) and CAP-H2 (bottom), only overlays are depicted. Representative images of single z planes are shown. Bars: (whole-cell diffraction-limited microscopy) 5 µm; (whole-cell STED microscopy) 1 µm; (zooms) 500 nm.

    Journal: The Journal of Cell Biology

    Article Title: A quantitative map of human Condensins provides new insights into mitotic chromosome architecture

    doi: 10.1083/jcb.201801048

    Figure Lengend Snippet: STED superresolution imaging of Condensins I and II. Condensin subunits were immunostained (anti-GFP), and mitotic cells with chromatids oriented in parallel to the focal plane were selected for imaging. DNA (Hoechst) and Condensins (mEGFP tag and anti-GFP immunostaining) were imaged by diffraction-limited microscopy, whereas immunostained Condensin was also imaged by STED microscopy. Representative images of mitotic chromatids with SMC4 (Condensin I + II), CAP-H (Condensin I), and CAP-H2 (Condensin II) in late prometaphase (left) and early anaphase (right) are shown. For SMC4 (top), whole-cell overview images as well as overlays of superresolved Condensin (white) and diffraction-limited DNA (magenta) imaging are shown. Overlays represent zooms into single chromatids (turquoise box). For CAP-H (middle) and CAP-H2 (bottom), only overlays are depicted. Representative images of single z planes are shown. Bars: (whole-cell diffraction-limited microscopy) 5 µm; (whole-cell STED microscopy) 1 µm; (zooms) 500 nm.

    Article Snippet: For tagging SMC4 at the C terminus and CAP-H at the N terminus with mEGFP, zinc finger nucleases (ZFNs) containing DNA binding sequences listed in Table S1 were purchased from Sigma-Aldrich.

    Techniques: Imaging, Immunostaining, Microscopy

    Force-extension traces for ( A ) the DNA-distamycin A complex; ( B ) the DNA complex with the α -helical peptide Ac-(Leu-Ala-Arg-Leu) 3 -NH-linker; ( C ) the complex of DNA with the 3 10 -helical peptide Ac-(Aib-Leu-Arg) 4 -NH-linker.

    Journal: Biophysical Journal

    Article Title: Identification of Binding Mechanisms in Single Molecule-DNA Complexes

    doi:

    Figure Lengend Snippet: Force-extension traces for ( A ) the DNA-distamycin A complex; ( B ) the DNA complex with the α -helical peptide Ac-(Leu-Ala-Arg-Leu) 3 -NH-linker; ( C ) the complex of DNA with the 3 10 -helical peptide Ac-(Aib-Leu-Arg) 4 -NH-linker.

    Article Snippet: The DNA-binding agents daunomycin (Sigma), ethidium bromide (Merck, Darmstadt, Germany), distamycin A (Sigma), YO (Molecular Probes, Eugene, OR), YOYO (Molecular Probes), Ac-(Leu-Ala-Arg-Leu)3 -NH-linker and Ac-(Aib-Leu-Arg)4 -NH-linker were added to 10 μ l of the DNA solution in a concentration of 150 μ M, corresponding to a 1:10 ratio of agent molecules per base pair.

    Techniques:

    Differential binding to the hsp70 heat shock element (HSE) maps to the heat shock transcription factor (HSF) DNA-binding domain. ( A ) DNase I footprinting by mHSF1 and mHSF2 to the hsp70 HSE. The hsp70 HSE probe was labeled at the 5′ end of the coding strand, and the concentration of the probe in all reaction mixtures was 0.1 nM. The amounts of bacterial lysates in lanes A–E were 0.5, 1, 2, 4, and 8 μL, respectively. Control reaction shows DNase I footprinting reaction in the absence of protein. The extent of HSF1 and HSF2 protection are indicated at the left with brackets. The positions of HSEs 1 to 5 are marked by arrows. ( B ) Specificity in DNA-binding maps to the DNA-binding domain. Chimeras in which the DBD was interchanged were expressed and purified as in Materials and Methods and used for DNase I footprinting. Lane assignments are as described for panel A . The extent of protein binding is delimited by the brackets.

    Journal: Genes & Development

    Article Title: The loop domain of heat shock transcription factor 1 dictates DNA-binding specificity and responses to heat stress

    doi: 10.1101/gad.894801

    Figure Lengend Snippet: Differential binding to the hsp70 heat shock element (HSE) maps to the heat shock transcription factor (HSF) DNA-binding domain. ( A ) DNase I footprinting by mHSF1 and mHSF2 to the hsp70 HSE. The hsp70 HSE probe was labeled at the 5′ end of the coding strand, and the concentration of the probe in all reaction mixtures was 0.1 nM. The amounts of bacterial lysates in lanes A–E were 0.5, 1, 2, 4, and 8 μL, respectively. Control reaction shows DNase I footprinting reaction in the absence of protein. The extent of HSF1 and HSF2 protection are indicated at the left with brackets. The positions of HSEs 1 to 5 are marked by arrows. ( B ) Specificity in DNA-binding maps to the DNA-binding domain. Chimeras in which the DBD was interchanged were expressed and purified as in Materials and Methods and used for DNase I footprinting. Lane assignments are as described for panel A . The extent of protein binding is delimited by the brackets.

    Article Snippet: To confirm that the His-tag on the carboxyl terminus did not affect the DNA-binding properties of the proteins, HSF1, HSF2, HSF1DB2, and HSF2DB1 were subcloned into the pET3 plasmid (Novagen) to yield constructs that did not carry His-tag.

    Techniques: Binding Assay, Footprinting, Labeling, Concentration Assay, Purification, Protein Binding

    Structure of the heat shock transcription factor (HSF) DNA-binding domain. ( A ) The central helix-turn-helix motif (red) is composed of α-helix2 and α-helix3, where the latter is the DNA recognition helix. The structure was modeled using the Ribbons program from the coordinates of NMR structure of the Drosophila ). The loop (yellow) was determined to be solvent exposed. ( B ) Alignment of the amino acid sequences for the mouse HSF1, HSF2, Drosophila HSF1, and Kluyveromyces lactis HSF DBDs. Predicted secondary structure for the mouse HSFs is based on homology to the solved K. lactis and Drosophila ), where cylinders represent α-helicies and arrows represent β-sheets. Residues conserved between HSF1 and HSF2 are boxed; residues identical among all four sequences are indicated by a solid oval; those positions that have conserved substitutions are indicated by an open oval. ( C ) Diagram of specific chimeras used in this study. HSF1 sequences (white); HSF2 sequences (black). (L) Linker; (HR A) trimerization domain.

    Journal: Genes & Development

    Article Title: The loop domain of heat shock transcription factor 1 dictates DNA-binding specificity and responses to heat stress

    doi: 10.1101/gad.894801

    Figure Lengend Snippet: Structure of the heat shock transcription factor (HSF) DNA-binding domain. ( A ) The central helix-turn-helix motif (red) is composed of α-helix2 and α-helix3, where the latter is the DNA recognition helix. The structure was modeled using the Ribbons program from the coordinates of NMR structure of the Drosophila ). The loop (yellow) was determined to be solvent exposed. ( B ) Alignment of the amino acid sequences for the mouse HSF1, HSF2, Drosophila HSF1, and Kluyveromyces lactis HSF DBDs. Predicted secondary structure for the mouse HSFs is based on homology to the solved K. lactis and Drosophila ), where cylinders represent α-helicies and arrows represent β-sheets. Residues conserved between HSF1 and HSF2 are boxed; residues identical among all four sequences are indicated by a solid oval; those positions that have conserved substitutions are indicated by an open oval. ( C ) Diagram of specific chimeras used in this study. HSF1 sequences (white); HSF2 sequences (black). (L) Linker; (HR A) trimerization domain.

    Article Snippet: To confirm that the His-tag on the carboxyl terminus did not affect the DNA-binding properties of the proteins, HSF1, HSF2, HSF1DB2, and HSF2DB1 were subcloned into the pET3 plasmid (Novagen) to yield constructs that did not carry His-tag.

    Techniques: Binding Assay, Nuclear Magnetic Resonance

    NF-κB, CREB, AP-1, and NF-IL-6 DNA binding activity in nuclear extracts of B16-F10 melanoma cells treated with corticosterone or NORA. Corticosterone ( C ) and NORA were incubated (at the concentrations indicated in Table 1 ) 18 h after seeding and were present in the incubation medium for 6 h. Nuclear extracts were then obtained as described under Materials and methods. Results are means + S.D. ( error bars ) of 4-5 independent experiments. The significance test refers to the comparison between each experimental condition versus controls (serum treated) (*P

    Journal: Journal of Translational Medicine

    Article Title: Stress hormones promote growth of B16-F10 melanoma metastases: an interleukin 6- and glutathione-dependent mechanism

    doi: 10.1186/1479-5876-11-72

    Figure Lengend Snippet: NF-κB, CREB, AP-1, and NF-IL-6 DNA binding activity in nuclear extracts of B16-F10 melanoma cells treated with corticosterone or NORA. Corticosterone ( C ) and NORA were incubated (at the concentrations indicated in Table 1 ) 18 h after seeding and were present in the incubation medium for 6 h. Nuclear extracts were then obtained as described under Materials and methods. Results are means + S.D. ( error bars ) of 4-5 independent experiments. The significance test refers to the comparison between each experimental condition versus controls (serum treated) (*P

    Article Snippet: DNA binding activity of NF-κB, CREB, AP-1, and NF-IL-6 Nuclear extracts were prepared with a nuclear extraction kit (Millipore, Billerica, MA).

    Techniques: Binding Assay, Activity Assay, Incubation