dnase i stock  (Millipore)


Bioz Verified Symbol Millipore is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    Deoxyribonuclease I
    Description:

    Catalog Number:
    d5793
    Price:
    None
    Buy from Supplier


    Structured Review

    Millipore dnase i stock
    Fig. 5. BRG1-dependent chromatin remodeling at the CIITA promoter. ( A ) <t>DNase</t> I accessibility requires BRG1. SW13 cells were infected with AdtTa plus AdBRG (lanes 2–7), or AdK798R (lanes 8–13). After 20 h, cells were left untreated (lanes 2–4 and 8–10) or exposed to IFN-γ for 24 h (lanes 5–7 and 11–13). Nuclei were harvested and treated with 12.8 (lanes 2, 5, 8 and 11), 25.6 (lanes 3, 6, 9 and 12) or 51.2 (lanes 4, 7, 10 and 13) ng/µl DNase I for 3 min at 37°C. DNA was isolated and analyzed by LM-PCR. ( B ) Hae III accessibility requires BRG1. Uninfected SW13 cells (lanes 2 and 3) or cells infected with AdtTa and AdBRG (lanes 4 and 5) were treated for 24 h with IFN-γ (lanes 2–5). Nuclei were isolated and incubated in vivo with Hae III (lanes 2 and 4) or no enzyme (lanes 3 and 5) at 37°C for 10 min (lanes 2 and 4). DNA was extracted, digested to completion in vitro with Avr II and analyzed by LM-PCR. The intensity of each Hae III band in lanes 2 and 4 was normalized to that of the full-length primer extension product to the Avr II site. The ratio of these values gave the fold increase in Hae III accessibility. Arrows on the left and right indicate the Hae III and Avr II sites, respectively. The marker in both (A) and (B) (lane 1) is Msp I-digested pBR322 DNA. The position of elements in the CIITA promoter is indicated to the right of each figure. Major (+1) and minor (+16) start sites of transcription are indicated.

    https://www.bioz.com/result/dnase i stock/product/Millipore
    Average 97 stars, based on 4976 article reviews
    Price from $9.99 to $1999.99
    dnase i stock - by Bioz Stars, 2020-09
    97/100 stars

    Images

    1) Product Images from "Interferon-?-induced chromatin remodeling at the CIITA locus is BRG1 dependent"

    Article Title: Interferon-?-induced chromatin remodeling at the CIITA locus is BRG1 dependent

    Journal: The EMBO Journal

    doi: 10.1093/emboj/21.8.1978

    Fig. 5. BRG1-dependent chromatin remodeling at the CIITA promoter. ( A ) DNase I accessibility requires BRG1. SW13 cells were infected with AdtTa plus AdBRG (lanes 2–7), or AdK798R (lanes 8–13). After 20 h, cells were left untreated (lanes 2–4 and 8–10) or exposed to IFN-γ for 24 h (lanes 5–7 and 11–13). Nuclei were harvested and treated with 12.8 (lanes 2, 5, 8 and 11), 25.6 (lanes 3, 6, 9 and 12) or 51.2 (lanes 4, 7, 10 and 13) ng/µl DNase I for 3 min at 37°C. DNA was isolated and analyzed by LM-PCR. ( B ) Hae III accessibility requires BRG1. Uninfected SW13 cells (lanes 2 and 3) or cells infected with AdtTa and AdBRG (lanes 4 and 5) were treated for 24 h with IFN-γ (lanes 2–5). Nuclei were isolated and incubated in vivo with Hae III (lanes 2 and 4) or no enzyme (lanes 3 and 5) at 37°C for 10 min (lanes 2 and 4). DNA was extracted, digested to completion in vitro with Avr II and analyzed by LM-PCR. The intensity of each Hae III band in lanes 2 and 4 was normalized to that of the full-length primer extension product to the Avr II site. The ratio of these values gave the fold increase in Hae III accessibility. Arrows on the left and right indicate the Hae III and Avr II sites, respectively. The marker in both (A) and (B) (lane 1) is Msp I-digested pBR322 DNA. The position of elements in the CIITA promoter is indicated to the right of each figure. Major (+1) and minor (+16) start sites of transcription are indicated.
    Figure Legend Snippet: Fig. 5. BRG1-dependent chromatin remodeling at the CIITA promoter. ( A ) DNase I accessibility requires BRG1. SW13 cells were infected with AdtTa plus AdBRG (lanes 2–7), or AdK798R (lanes 8–13). After 20 h, cells were left untreated (lanes 2–4 and 8–10) or exposed to IFN-γ for 24 h (lanes 5–7 and 11–13). Nuclei were harvested and treated with 12.8 (lanes 2, 5, 8 and 11), 25.6 (lanes 3, 6, 9 and 12) or 51.2 (lanes 4, 7, 10 and 13) ng/µl DNase I for 3 min at 37°C. DNA was isolated and analyzed by LM-PCR. ( B ) Hae III accessibility requires BRG1. Uninfected SW13 cells (lanes 2 and 3) or cells infected with AdtTa and AdBRG (lanes 4 and 5) were treated for 24 h with IFN-γ (lanes 2–5). Nuclei were isolated and incubated in vivo with Hae III (lanes 2 and 4) or no enzyme (lanes 3 and 5) at 37°C for 10 min (lanes 2 and 4). DNA was extracted, digested to completion in vitro with Avr II and analyzed by LM-PCR. The intensity of each Hae III band in lanes 2 and 4 was normalized to that of the full-length primer extension product to the Avr II site. The ratio of these values gave the fold increase in Hae III accessibility. Arrows on the left and right indicate the Hae III and Avr II sites, respectively. The marker in both (A) and (B) (lane 1) is Msp I-digested pBR322 DNA. The position of elements in the CIITA promoter is indicated to the right of each figure. Major (+1) and minor (+16) start sites of transcription are indicated.

    Techniques Used: Infection, Isolation, Polymerase Chain Reaction, Incubation, In Vivo, In Vitro, Marker

    2) Product Images from "Transcriptional Regulation of the phoPR Operon in Bacillus subtilis"

    Article Title: Transcriptional Regulation of the phoPR Operon in Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.4.1182-1190.2004

    DNase I footprinting assay of the ykoL promoter using PhoP and PhoP∼P. A PCR fragment corresponding to the ykoL promoter region (−153 to +60 relative to the transcriptional start site of ykoL ) was used as a DNA probe. Coding strand footprinting 32 P-labeled YkoL-FOR primer and the noncoding strand footprinting 32 P-labeled YkoL-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The black vertical lines show the regions that were bound by PhoP and PhoP∼P. The thick black lines show the regions where PhoP and PhoP∼P bound with a higher affinity. The numbers indicate the positions of the PhoP binding sites relative to the transcriptional start site M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the ykoL promoter the translational start site, RBS, transcriptional start site (+1), and corresponding −35 and −10 sequences of the promoter are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of PhoP dimer (5 ± 2-bp spacer and a maximum of two mismatches). The positions of YkoL-FOR and YkoL-REV primers are shown by thick arrows and are labeled.
    Figure Legend Snippet: DNase I footprinting assay of the ykoL promoter using PhoP and PhoP∼P. A PCR fragment corresponding to the ykoL promoter region (−153 to +60 relative to the transcriptional start site of ykoL ) was used as a DNA probe. Coding strand footprinting 32 P-labeled YkoL-FOR primer and the noncoding strand footprinting 32 P-labeled YkoL-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The black vertical lines show the regions that were bound by PhoP and PhoP∼P. The thick black lines show the regions where PhoP and PhoP∼P bound with a higher affinity. The numbers indicate the positions of the PhoP binding sites relative to the transcriptional start site M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the ykoL promoter the translational start site, RBS, transcriptional start site (+1), and corresponding −35 and −10 sequences of the promoter are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of PhoP dimer (5 ± 2-bp spacer and a maximum of two mismatches). The positions of YkoL-FOR and YkoL-REV primers are shown by thick arrows and are labeled.

    Techniques Used: Footprinting, Polymerase Chain Reaction, Labeling, Incubation, Binding Assay, Sequencing

    DNase I footprinting assay of the phoPR promoter by using PhoP and PhoP∼P. A PCR fragment corresponding to the phoPR promoter region (−205 to +16 relative to the translational start site of phoP ) was used as the DNA probe. Coding strand footprinting 32 P-labeled PhoP-FOR primer and the noncoding strand footprinting 32 P-labeled PhoP-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The thick black vertical lines show the regions where PhoP and PhoP∼P bound. The numbers indicate the positions of the PhoP binding sites relative to the translational start site. M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the phoPR promoter (lower part of the figure), the translational start site, ribosome binding site (RBS), transcriptional start sites (TS), and corresponding −35 and −10 sequences of the P 1 and P 2 promoters are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of the PhoP dimer (5 ± 2-bp spacer and maximum of two mismatches). The positions of PhoP-FOR and PhoP-REV primers are shown by thick arrows and are labeled.
    Figure Legend Snippet: DNase I footprinting assay of the phoPR promoter by using PhoP and PhoP∼P. A PCR fragment corresponding to the phoPR promoter region (−205 to +16 relative to the translational start site of phoP ) was used as the DNA probe. Coding strand footprinting 32 P-labeled PhoP-FOR primer and the noncoding strand footprinting 32 P-labeled PhoP-REV primer were used in the PCR to prepare the DNA probes. Increasing amounts (0, 19, 38, 57, and 76 μM; lanes 1 to 5) of PhoP were incubated with PhoR231 (4 μM) in the presence or absence of ATP and were mixed with the DNA probe. The thick black vertical lines show the regions where PhoP and PhoP∼P bound. The numbers indicate the positions of the PhoP binding sites relative to the translational start site. M is the A+G Maxam and Gilbert sequencing reaction lane used as size markers. In the sequence of the phoPR promoter (lower part of the figure), the translational start site, ribosome binding site (RBS), transcriptional start sites (TS), and corresponding −35 and −10 sequences of the P 1 and P 2 promoters are underlined and labeled. Grey shading indicates direct repeats of TT(A/T/C)ACA for putative binding of the PhoP dimer (5 ± 2-bp spacer and maximum of two mismatches). The positions of PhoP-FOR and PhoP-REV primers are shown by thick arrows and are labeled.

    Techniques Used: Footprinting, Polymerase Chain Reaction, Labeling, Incubation, Binding Assay, Sequencing

    3) Product Images from "Subtelomeric proteins negatively regulate telomere elongation in budding yeast"

    Article Title: Subtelomeric proteins negatively regulate telomere elongation in budding yeast

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600975

    Structure of the telomeric end-complex and subtelomeric chromatin in TEL1 and tel1 Δ cells. ( A ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a TG 1–3 probe. ( B ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a Y′ probe. ( C ) Analysis of the profiles of the TG 1–3 hybridizing DNA. The thick line represents the tel1 Δ samples. The thin line represents the TEL1 samples.
    Figure Legend Snippet: Structure of the telomeric end-complex and subtelomeric chromatin in TEL1 and tel1 Δ cells. ( A ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a TG 1–3 probe. ( B ) DNA was isolated from fresh cell lysates treated with DNase I or MNase and then analyzed by Southern blot with a Y′ probe. ( C ) Analysis of the profiles of the TG 1–3 hybridizing DNA. The thick line represents the tel1 Δ samples. The thin line represents the TEL1 samples.

    Techniques Used: Isolation, Southern Blot

    4) Product Images from "Pathogenic Profiles and Molecular Signatures of Antinuclear Autoantibodies Rescued from NZM2410 Lupus Mice"

    Article Title: Pathogenic Profiles and Molecular Signatures of Antinuclear Autoantibodies Rescued from NZM2410 Lupus Mice

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20030132

    Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).
    Figure Legend Snippet: Nuclear antigenic bridges facilitate histone binding by anti-DNA Abs. Six representative anti-dsDNA/antihistone dual-binding Abs were subjected to DNase-I or sham treatment as detailed in Materials and Methods. Likewise, the histone substrate (i.e., “Antigen”) was also subjected to DNase-I or sham treatment. All Abs were tested for histone reactivity within the same ELISA plates. Horizontal bars represent the mean histone reactivity within each treatment group. The depicted p-values represent the result of comparing each group with the sham-treated (Ab and Ag) control. All DNase-I–treated Abs retained dsDNA-binding after treatment (not depicted).

    Techniques Used: Binding Assay, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections"

    Article Title: Haemophilus parainfluenzae Strain ATCC 33392 Forms Biofilms In Vitro and during Experimental Otitis Media Infections

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01070-16

    H. parainfluenzae biofilms are susceptible to detachment by DNase I and proteinase K (PK). Bacterial biofilms formed in 96-well plates were washed and incubated with different concentrations of DNase I (A) or PK (B) in enzyme buffer for 1 h at 37°C. Biofilms were quantitated by crystal violet staining as described in Materials and Methods. Bars represent the mean absorbance. Error bars show the standard deviations. Asterisks indicate significant decreases in biofilm biomass following enzymatic treatment compared to the untreated biofilm biomass. P values were determined by Student's t test. *, P
    Figure Legend Snippet: H. parainfluenzae biofilms are susceptible to detachment by DNase I and proteinase K (PK). Bacterial biofilms formed in 96-well plates were washed and incubated with different concentrations of DNase I (A) or PK (B) in enzyme buffer for 1 h at 37°C. Biofilms were quantitated by crystal violet staining as described in Materials and Methods. Bars represent the mean absorbance. Error bars show the standard deviations. Asterisks indicate significant decreases in biofilm biomass following enzymatic treatment compared to the untreated biofilm biomass. P values were determined by Student's t test. *, P

    Techniques Used: Incubation, Staining

    DNase I and proteinase K (PK) inhibit H. parainfluenzae biofilm formation. Bacteria were cultured with NBHI medium or NBHI medium supplemented with DNase I (1 mg ml −1 ) or proteinase K (1 mg ml −1 ) in 96-well plates, 24-well plates, or 4-well chamber slides for 24 h. (A) Biofilms formed in 96-well plates were washed and quantitated by crystal violet staining for measuring biofilm biomass. (B) Biofilms formed in 24-well plates were serially diluted and plated for viable bacterial counts. Biofilms formed in 4-well chamber slides were stained with Syto 9 alone for labeling all biofilm cells and visualized by CLSM. (C to E) Z-series images were used to create representative volumetric views of untreated (C), DNase I-treated (D), and proteinase K-treated (E) biofilms. (F to H) Z-series images were also exported to COMSTAT to obtain biofilm measurements, including average biofilm thickness (F), total biomass (G), and surface roughness coefficient (H). Error bars indicate standard deviations. Asterisks indicate a significant difference compared to the untreated biofilm as determined by Student's t test. **, P
    Figure Legend Snippet: DNase I and proteinase K (PK) inhibit H. parainfluenzae biofilm formation. Bacteria were cultured with NBHI medium or NBHI medium supplemented with DNase I (1 mg ml −1 ) or proteinase K (1 mg ml −1 ) in 96-well plates, 24-well plates, or 4-well chamber slides for 24 h. (A) Biofilms formed in 96-well plates were washed and quantitated by crystal violet staining for measuring biofilm biomass. (B) Biofilms formed in 24-well plates were serially diluted and plated for viable bacterial counts. Biofilms formed in 4-well chamber slides were stained with Syto 9 alone for labeling all biofilm cells and visualized by CLSM. (C to E) Z-series images were used to create representative volumetric views of untreated (C), DNase I-treated (D), and proteinase K-treated (E) biofilms. (F to H) Z-series images were also exported to COMSTAT to obtain biofilm measurements, including average biofilm thickness (F), total biomass (G), and surface roughness coefficient (H). Error bars indicate standard deviations. Asterisks indicate a significant difference compared to the untreated biofilm as determined by Student's t test. **, P

    Techniques Used: Cell Culture, Staining, Labeling, Confocal Laser Scanning Microscopy

    6) Product Images from "Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2"

    Article Title: Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2

    Journal: Nature Communications

    doi: 10.1038/s41467-017-01788-7

    Pcid2 deficiency causes H2A.Z deposition to lymphoid fate regulator genes in MPPs. a Sorted MPPs were lysed for ChIP assays. Indicated promoters were examined by qPCR. Signals were normalized to input DNA. Fold enrichment was calculated comparing with negative control (Non-pro locus). Results are shown as means ± S.D. b , c Indicated MPPs were lysed for ChIP assays with anti-H2A b or anti-SRCAP c antibody as in a . d MPP lysates were incubated with anti-SRCAP antibody. SRCAP-associated PU.1 and GATA1 were detected by immunoblotting. IP immunoprecipitation, RIgG rabbit IgG. e Indicated MPPs were lysed for ChIP assays with anti-PU.1 antibody as in a . f Expression levels of lymphoid fate regulator genes and myeloid fate regulator genes were assessed in sorted MPPs by quantitative RT-PCR. g Nuclei of MPPs were extracted for DNase I digestion assay. Chromatin accessibility were quantitated by qPCR. h Flag-PU.1, pTK and pGL3- Il7r promoter or pGL3- Ikzf1 promoter, together with the indicated shRNAs, were transfected into 293T cells for luciferase assays. Results are shown as means ± S.D. i Pcid2 and Spi1 DKO were generated as described in Methods section. Bone marrow mRNA levels of Pcid2 and Spi1 were measured by qPCR. j In vitro lymphoid differentiation assays of the indicated LMPPs were conducted as in Fig. 2c . For Pcid2 or PU.1 restoration, 1 × 10 3 HSCs were isolated from the BM of Spi1 −/− or Pcid2 −/− mice and infected with pMY-GFP-Pcid2 or pMY-GFP-PU.1 containing retrovirus and transplanted into lethally irradiated recipient mice together with 5 × 10 6 helper cells. k Cells were sorted, infected and cultured as in j and used Flt3 ligand alone thereafter. l B-cell and myeloid differentiation assays of the indicated MPPs in vitro were conducted as in Fig. 2e . m Indicated HSCs as described in j were sorted, infected and transplanted for 16 weeks. m Percentages of MPPs, n CLPs and o CMPs from the BM of indicated mice were analysed by flow cytometry. Results are shown as means ± S.D. n = 6 for each group. ** P
    Figure Legend Snippet: Pcid2 deficiency causes H2A.Z deposition to lymphoid fate regulator genes in MPPs. a Sorted MPPs were lysed for ChIP assays. Indicated promoters were examined by qPCR. Signals were normalized to input DNA. Fold enrichment was calculated comparing with negative control (Non-pro locus). Results are shown as means ± S.D. b , c Indicated MPPs were lysed for ChIP assays with anti-H2A b or anti-SRCAP c antibody as in a . d MPP lysates were incubated with anti-SRCAP antibody. SRCAP-associated PU.1 and GATA1 were detected by immunoblotting. IP immunoprecipitation, RIgG rabbit IgG. e Indicated MPPs were lysed for ChIP assays with anti-PU.1 antibody as in a . f Expression levels of lymphoid fate regulator genes and myeloid fate regulator genes were assessed in sorted MPPs by quantitative RT-PCR. g Nuclei of MPPs were extracted for DNase I digestion assay. Chromatin accessibility were quantitated by qPCR. h Flag-PU.1, pTK and pGL3- Il7r promoter or pGL3- Ikzf1 promoter, together with the indicated shRNAs, were transfected into 293T cells for luciferase assays. Results are shown as means ± S.D. i Pcid2 and Spi1 DKO were generated as described in Methods section. Bone marrow mRNA levels of Pcid2 and Spi1 were measured by qPCR. j In vitro lymphoid differentiation assays of the indicated LMPPs were conducted as in Fig. 2c . For Pcid2 or PU.1 restoration, 1 × 10 3 HSCs were isolated from the BM of Spi1 −/− or Pcid2 −/− mice and infected with pMY-GFP-Pcid2 or pMY-GFP-PU.1 containing retrovirus and transplanted into lethally irradiated recipient mice together with 5 × 10 6 helper cells. k Cells were sorted, infected and cultured as in j and used Flt3 ligand alone thereafter. l B-cell and myeloid differentiation assays of the indicated MPPs in vitro were conducted as in Fig. 2e . m Indicated HSCs as described in j were sorted, infected and transplanted for 16 weeks. m Percentages of MPPs, n CLPs and o CMPs from the BM of indicated mice were analysed by flow cytometry. Results are shown as means ± S.D. n = 6 for each group. ** P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Incubation, Immunoprecipitation, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Generated, In Vitro, Isolation, Mouse Assay, Infection, Irradiation, Cell Culture, Flow Cytometry, Cytometry

    7) Product Images from "Intercalation of calcein into layered silicate magadiite and their optical properties"

    Article Title: Intercalation of calcein into layered silicate magadiite and their optical properties

    Journal: Royal Society Open Science

    doi: 10.1098/rsos.171258

    Elemental mapping images of Ca-magadiite–calcein ( a – f ), Zn-magadiite–calcein ( g – l ) and Al-magadiite–calcein ( m – r ).
    Figure Legend Snippet: Elemental mapping images of Ca-magadiite–calcein ( a – f ), Zn-magadiite–calcein ( g – l ) and Al-magadiite–calcein ( m – r ).

    Techniques Used:

    ( a – c ) FTIR spectra of magadiite, H-magadiite, calcein, Ca, Zn, Al ion-exchanged magadiites and their intercalated compounds; ( d ) Comparative FTIR spectra of calcein, Ca-magadiite–calcein, Zn-magadiite–calcein and Al-magadiite–calcein.
    Figure Legend Snippet: ( a – c ) FTIR spectra of magadiite, H-magadiite, calcein, Ca, Zn, Al ion-exchanged magadiites and their intercalated compounds; ( d ) Comparative FTIR spectra of calcein, Ca-magadiite–calcein, Zn-magadiite–calcein and Al-magadiite–calcein.

    Techniques Used:

    ( a – c ) XRD patterns of magadiite, H-magadiite, calcein, Ca, Zn, Al ion-exchanged magadiites and their intercalated compounds.
    Figure Legend Snippet: ( a – c ) XRD patterns of magadiite, H-magadiite, calcein, Ca, Zn, Al ion-exchanged magadiites and their intercalated compounds.

    Techniques Used:

    Inverted fluorescence microscope images of Ca-magadiite–calcein ( a ), Zn-magadiite–calcein ( b ) and Al-magadiite–calcein ( c ) under 250 nm UV light irradiation.
    Figure Legend Snippet: Inverted fluorescence microscope images of Ca-magadiite–calcein ( a ), Zn-magadiite–calcein ( b ) and Al-magadiite–calcein ( c ) under 250 nm UV light irradiation.

    Techniques Used: Fluorescence, Microscopy, Irradiation

    XPS spectra of Ca-magadiite–calcein ( a ), Zn-magadiite–calcein ( b ) and Al-magadiite–calcein ( c ); inserting core-level spectra of Ca 2p , Zn 2p , Al 2p .
    Figure Legend Snippet: XPS spectra of Ca-magadiite–calcein ( a ), Zn-magadiite–calcein ( b ) and Al-magadiite–calcein ( c ); inserting core-level spectra of Ca 2p , Zn 2p , Al 2p .

    Techniques Used:

    A schematic illustration of synthesis of intercalation of calcein into the interlayer spaces of Ca-, Zn-, Al-magadiites (Ca-magadiite–calcein, Zn-magadiite–calcein and Al-magadiite–calcein).
    Figure Legend Snippet: A schematic illustration of synthesis of intercalation of calcein into the interlayer spaces of Ca-, Zn-, Al-magadiites (Ca-magadiite–calcein, Zn-magadiite–calcein and Al-magadiite–calcein).

    Techniques Used:

    Molecular structure of calcein.
    Figure Legend Snippet: Molecular structure of calcein.

    Techniques Used:

    Fluorescence spectra of calcein, Ca-magadiite–calcein, Zn-magadiite–calcein and Al-magadiite–calcein.
    Figure Legend Snippet: Fluorescence spectra of calcein, Ca-magadiite–calcein, Zn-magadiite–calcein and Al-magadiite–calcein.

    Techniques Used: Fluorescence

    TG curves of ( a ) Ca-magadiite and Ca-magadiite–calcein, ( b ) Zn-magadiite and Zn-magadiite–calcein and ( c ) Al-magadiite and Al-magadiite–calcein; inserting their corresponding DTA curves.
    Figure Legend Snippet: TG curves of ( a ) Ca-magadiite and Ca-magadiite–calcein, ( b ) Zn-magadiite and Zn-magadiite–calcein and ( c ) Al-magadiite and Al-magadiite–calcein; inserting their corresponding DTA curves.

    Techniques Used:

    SEM images of Ca-magadiite–calcein ( a , b ), Zn-magadiite–calcein ( c , d ) and Al-magadiite–calcein ( e , f ).
    Figure Legend Snippet: SEM images of Ca-magadiite–calcein ( a , b ), Zn-magadiite–calcein ( c , d ) and Al-magadiite–calcein ( e , f ).

    Techniques Used:

    8) Product Images from "Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-?B-dependent pathways"

    Article Title: Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2- and NF-?B-dependent pathways

    Journal: British Journal of Pharmacology

    doi: 10.1111/j.1476-5381.2012.01905.x

    Western blot analysis of p-IκB-α and p-ERK1/2 protein levels from lungs of PBS-challenged mice pretreated with vehicle (PBS), from ovalbumin (OVA)-challenged mice pretreated with vehicle (OVA), Ang-(1–7) and A779 plus Ang-(1–7).
    Figure Legend Snippet: Western blot analysis of p-IκB-α and p-ERK1/2 protein levels from lungs of PBS-challenged mice pretreated with vehicle (PBS), from ovalbumin (OVA)-challenged mice pretreated with vehicle (OVA), Ang-(1–7) and A779 plus Ang-(1–7).

    Techniques Used: Western Blot, Mouse Assay

    Histological examination of PAS stain of ovalbumin-challenged/vehicle-pretreated mice shows significant bronchial mucus production and goblet cell hyper/metaplasia in mice (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7)
    Figure Legend Snippet: Histological examination of PAS stain of ovalbumin-challenged/vehicle-pretreated mice shows significant bronchial mucus production and goblet cell hyper/metaplasia in mice (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7)

    Techniques Used: Staining, Mouse Assay

    Masson's Trichrome staining of lung samples from ovalbumin-challenged/vehicle-treated mice show significant peribronchial and perivascular fibrosis (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7) (0.3 mg·kg
    Figure Legend Snippet: Masson's Trichrome staining of lung samples from ovalbumin-challenged/vehicle-treated mice show significant peribronchial and perivascular fibrosis (B) ( n = 6) compared with PBS-challenged mice (A) ( n = 6). Treatment with Ang-(1–7) (0.3 mg·kg

    Techniques Used: Staining, Mouse Assay

    Representative low-magnification light photomicrographs display H E staining of whole lung samples from (A) PBS vehicle ( n = 6), (B) ovalbumin (OVA)-challenged ( n = 6), (C) OVA-challenged, Ang-(1–7) (0.3 mg·kg −1 ; i.p.)
    Figure Legend Snippet: Representative low-magnification light photomicrographs display H E staining of whole lung samples from (A) PBS vehicle ( n = 6), (B) ovalbumin (OVA)-challenged ( n = 6), (C) OVA-challenged, Ang-(1–7) (0.3 mg·kg −1 ; i.p.)

    Techniques Used: Staining

    9) Product Images from "An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells"

    Article Title: An interplay between extracellular signalling and the dynamics of the exit from pluripotency drives cell fate decisions in mouse ES cells

    Journal: Biology Open

    doi: 10.1242/bio.20148409

    The exit from pluripotency determines the ability of mESCs to differentiate. (A) Sox1::GFP (left) or T::GFP (right) mESCs exposed to serum and LIF (SL), 2i and LIF (2i + L), N2B27 or Activin and Chi (AC) for the indicated durations and GFP expression analysed by flow cytometry. Hashed vertical line bisecting the population profile plots indicates the peak maximum of the negative control. (B) T::GFP mESCs differentiated in AC for 2 days (0–2), immunostained for Hoechst, Oct4 and Sox2 and imaged by confocal microscopy. (C) TNGA mESCs cultured in SL and FACS sorted into low- (indicated in pink) and high- (indicated in dark blue) expressing populations (top) were replated in AC conditions for 2 days, immunostained for Brachyury and analysed by confocal microscopy (bottom). TNGA cells cultured in SL conditions served as a negative control for Brachyury immunostaining. (D) T::GFP (red) and Sox1::GFP (blue) mESCs were differentiated for 2 days in AC conditions after exposure to N2B27 for the indicated durations. GFP expression was measured by flow cytometry. (E) T::GFP (Red) or Sox1::GFP (black) mESCs plated and treated with N2B27 for 6 days with single, 1-day pulses of AC on days indicated above graphs. For the control, T::GFP or Sox1::GFP cells were incubated with 6 days of either AC or N2B27, respectively. Grey bar indicates period of AC pulsing. (F) Sox1::GFP mESCs treated with 1 ng/ml BMP4 for durations indicated or 5 days (0–5) in N2B27. GFP expression measured by flow cytometry and fluorescence values displayed are normalised to the N2B27 control. (G,H) Live-cell imaging of TNGA cells in N2B27 alone (G) or supplemented with 1 ng/ml BMP4 (H). Scale bars: 100 µm.
    Figure Legend Snippet: The exit from pluripotency determines the ability of mESCs to differentiate. (A) Sox1::GFP (left) or T::GFP (right) mESCs exposed to serum and LIF (SL), 2i and LIF (2i + L), N2B27 or Activin and Chi (AC) for the indicated durations and GFP expression analysed by flow cytometry. Hashed vertical line bisecting the population profile plots indicates the peak maximum of the negative control. (B) T::GFP mESCs differentiated in AC for 2 days (0–2), immunostained for Hoechst, Oct4 and Sox2 and imaged by confocal microscopy. (C) TNGA mESCs cultured in SL and FACS sorted into low- (indicated in pink) and high- (indicated in dark blue) expressing populations (top) were replated in AC conditions for 2 days, immunostained for Brachyury and analysed by confocal microscopy (bottom). TNGA cells cultured in SL conditions served as a negative control for Brachyury immunostaining. (D) T::GFP (red) and Sox1::GFP (blue) mESCs were differentiated for 2 days in AC conditions after exposure to N2B27 for the indicated durations. GFP expression was measured by flow cytometry. (E) T::GFP (Red) or Sox1::GFP (black) mESCs plated and treated with N2B27 for 6 days with single, 1-day pulses of AC on days indicated above graphs. For the control, T::GFP or Sox1::GFP cells were incubated with 6 days of either AC or N2B27, respectively. Grey bar indicates period of AC pulsing. (F) Sox1::GFP mESCs treated with 1 ng/ml BMP4 for durations indicated or 5 days (0–5) in N2B27. GFP expression measured by flow cytometry and fluorescence values displayed are normalised to the N2B27 control. (G,H) Live-cell imaging of TNGA cells in N2B27 alone (G) or supplemented with 1 ng/ml BMP4 (H). Scale bars: 100 µm.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Negative Control, Confocal Microscopy, Cell Culture, FACS, Immunostaining, Incubation, Fluorescence, Live Cell Imaging

    10) Product Images from "Top-Down Proteomic Identification of Furin-Cleaved α-Subunit of Shiga Toxin 2 from Escherichia coli O157:H7 Using MALDI-TOF-TOF-MS/MS"

    Article Title: Top-Down Proteomic Identification of Furin-Cleaved α-Subunit of Shiga Toxin 2 from Escherichia coli O157:H7 Using MALDI-TOF-TOF-MS/MS

    Journal: Journal of Biomedicine and Biotechnology

    doi: 10.1155/2010/123460

    Bottom-up proteomic sequence coverage of α -Stx2 (in red) from select gel bands (Figure S3, Supplementary Materials). A 22-residue signal peptide (in bold) is removed in the mature protein. Recognition sites for potential furin cleavage (RXXR) are boxed. An asterisk (∗) marks the only observed furin cleavage site.
    Figure Legend Snippet: Bottom-up proteomic sequence coverage of α -Stx2 (in red) from select gel bands (Figure S3, Supplementary Materials). A 22-residue signal peptide (in bold) is removed in the mature protein. Recognition sites for potential furin cleavage (RXXR) are boxed. An asterisk (∗) marks the only observed furin cleavage site.

    Techniques Used: Sequencing

    Sequence of α -Stx2 from E. coli O157:H7 (EDL933). A 22-residue signal peptide (in bold) is removed in the mature protein, and cysteines involved in an intramolecular disulfide bond ( S ⋯ S ) are boxed. Potential furin cleavage recognition sites (RXXR) are boxed. An asterisk (∗) marks the observed furin cleavage site. In eukaryotic cells, after disulfide reduction, the catalytically active α A1 -Stx2 fragment is translocated to the cytosol and the α A2 -Stx2 fragment remains associated with the β -pentamer.
    Figure Legend Snippet: Sequence of α -Stx2 from E. coli O157:H7 (EDL933). A 22-residue signal peptide (in bold) is removed in the mature protein, and cysteines involved in an intramolecular disulfide bond ( S ⋯ S ) are boxed. Potential furin cleavage recognition sites (RXXR) are boxed. An asterisk (∗) marks the observed furin cleavage site. In eukaryotic cells, after disulfide reduction, the catalytically active α A1 -Stx2 fragment is translocated to the cytosol and the α A2 -Stx2 fragment remains associated with the β -pentamer.

    Techniques Used: Sequencing

    11) Product Images from "High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells"

    Article Title: High Throughput NPY-Venus and Serotonin Secretion Assays for Regulated Exocytosis in Neuroendocrine Cells

    Journal: Bio-protocol

    doi: 10.21769/BioProtoc.2680

    A graphic summary of the NPY-Venus secretion assay A. Plate design for an example experiment of testing two siRNAs of interest. A positive control siRNA that is known to inhibit NPY-Venus secretion ( e.g. ,  CADPS  siRNA) and a negative control siRNA that is known not to affect NPY-venus secretion ( e.g. , Dharmacon non-targeting siRNA D-001210-03-20) should always be included in each experiment. 8 wells are used for each siRNA with 4 wells for stimulation with ionomycin/Ca 2+  and the other 4 for background secretion. 6 wells are left blank so that the same wells on the black assay plates (see B) can be used for determining the background fluorescence of plain buffer. pos control siRNA: positive control siRNA; neg control siRNA: negative control siRNA. B. Steps of the NPY-Venus secretion assay. From left to right: conduct reverse transfection with siRNAs; after two days of incubation, remove culture medium and wash cells with pre-warmed PSS-Na; stimulate cells with PSS-Na containing either ionomycin or DMSO and then transfer the supernatant to a black assay plate; lyse cells with 1% Triton X-100 and then transfer to a black assay plate; add plain buffer to the reserved wells (see A) and then determine NPY-Venus fluorescence with a plate reader. Refer to texts for details.
    Figure Legend Snippet: A graphic summary of the NPY-Venus secretion assay A. Plate design for an example experiment of testing two siRNAs of interest. A positive control siRNA that is known to inhibit NPY-Venus secretion ( e.g. , CADPS siRNA) and a negative control siRNA that is known not to affect NPY-venus secretion ( e.g. , Dharmacon non-targeting siRNA D-001210-03-20) should always be included in each experiment. 8 wells are used for each siRNA with 4 wells for stimulation with ionomycin/Ca 2+ and the other 4 for background secretion. 6 wells are left blank so that the same wells on the black assay plates (see B) can be used for determining the background fluorescence of plain buffer. pos control siRNA: positive control siRNA; neg control siRNA: negative control siRNA. B. Steps of the NPY-Venus secretion assay. From left to right: conduct reverse transfection with siRNAs; after two days of incubation, remove culture medium and wash cells with pre-warmed PSS-Na; stimulate cells with PSS-Na containing either ionomycin or DMSO and then transfer the supernatant to a black assay plate; lyse cells with 1% Triton X-100 and then transfer to a black assay plate; add plain buffer to the reserved wells (see A) and then determine NPY-Venus fluorescence with a plate reader. Refer to texts for details.

    Techniques Used: Positive Control, Negative Control, Fluorescence, Transfection, Incubation

    12) Product Images from "Identification and Functional Characterization of CbaR, a MarR-Like Modulator of the cbaABC-Encoded Chlorobenzoate Catabolism Pathway"

    Article Title: Identification and Functional Characterization of CbaR, a MarR-Like Modulator of the cbaABC-Encoded Chlorobenzoate Catabolism Pathway

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.67.8.3530-3541.2001

    (A) DNase I protection assay to determine specific regions of P cbaA DNA protected by CbaR. Template DNA was end labeled with 32 PO 4 on the noncoding strand ( 32 P-PCBA) or the coding strand ( 32 P-CBAA). Various amounts of CbaR were added, and the effect of 4 mM 3CBA (3Cba) or 4 mM 3-carboxybenzoate (3Crba) on CbaR protection of two regions (sites I and II) was determined. A sequencing reaction conducted with the appropriate primers was included during electrophoretic resolution of DNase I-digested samples. (B) Sequences of both strands of the template DNA used for the assay, indicating the positions of sites I and II relative to the transcriptional start site of cbaA (position +1); the putative −10 hexamer; and the start codon of cbaA (ATG). Inverted repeats found in site I and modified versions of these repeats in site II are indicated by arrows, and bases present in both sites are indicated by boldface type.
    Figure Legend Snippet: (A) DNase I protection assay to determine specific regions of P cbaA DNA protected by CbaR. Template DNA was end labeled with 32 PO 4 on the noncoding strand ( 32 P-PCBA) or the coding strand ( 32 P-CBAA). Various amounts of CbaR were added, and the effect of 4 mM 3CBA (3Cba) or 4 mM 3-carboxybenzoate (3Crba) on CbaR protection of two regions (sites I and II) was determined. A sequencing reaction conducted with the appropriate primers was included during electrophoretic resolution of DNase I-digested samples. (B) Sequences of both strands of the template DNA used for the assay, indicating the positions of sites I and II relative to the transcriptional start site of cbaA (position +1); the putative −10 hexamer; and the start codon of cbaA (ATG). Inverted repeats found in site I and modified versions of these repeats in site II are indicated by arrows, and bases present in both sites are indicated by boldface type.

    Techniques Used: Labeling, Sequencing, Modification

    13) Product Images from "A novel role for synaptic acetylcholinesterase as an apoptotic deoxyribonuclease"

    Article Title: A novel role for synaptic acetylcholinesterase as an apoptotic deoxyribonuclease

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2015.2

    Interaction between synaptic acetylcholinesterase (AChE S ) polypeptide with plasmid DNA. ( a ) Silver staining and western blot showing the purified human AChE S polypeptide, hAChE-T547. The anti-AChE antibody provided by Dr Palmer Taylor was used at a dilution of 1:1000. ( b ) Cholinesterase activity of hAChE-T547 examined by the Ellman assay, with measurement of optical density (OD) 405 nm. Data shown represent one of three independent experiments. Values represent the mean±s.d. of triplicate samples. ( c ) The interaction between hAChE-T547 and pEGFP-c1 plasmid DNA examined by Biacore T100. DNase I and bovine serum albumin (BSA) were used as positive and negative controls, respectively. Data shown represent one of three independent experiments.
    Figure Legend Snippet: Interaction between synaptic acetylcholinesterase (AChE S ) polypeptide with plasmid DNA. ( a ) Silver staining and western blot showing the purified human AChE S polypeptide, hAChE-T547. The anti-AChE antibody provided by Dr Palmer Taylor was used at a dilution of 1:1000. ( b ) Cholinesterase activity of hAChE-T547 examined by the Ellman assay, with measurement of optical density (OD) 405 nm. Data shown represent one of three independent experiments. Values represent the mean±s.d. of triplicate samples. ( c ) The interaction between hAChE-T547 and pEGFP-c1 plasmid DNA examined by Biacore T100. DNase I and bovine serum albumin (BSA) were used as positive and negative controls, respectively. Data shown represent one of three independent experiments.

    Techniques Used: Plasmid Preparation, Silver Staining, Western Blot, Purification, Activity Assay

    Purified synaptic acetylcholinesterase polypeptide cleaves DNA in a cell-free hydrolysis system. ( a ) Agarose gel images showing pEGFP-c1 plasmid DNA (150 ng) incubated with purified hAChE-T547 at the indicated concentration at 37 °C for 6 h. Eco RI-digested DNA (0.5 U/μl) was used to demarcate the linearized plasmid. Bovine serum albumin (BSA) was used as a negative control. ( b ) Agarose gel images of pEGFP-c1 plasmid DNA (150 ng) incubated with 0.5 μ M hAChE-T547 at 37 °C for the indicated time periods. ( c ) Agarose gel images of pEGFP-c1 plasmid DNA (150–600 ng) incubated with or without 0.5 μ M hAChE-T547 at 37 °C for 6 h. ( d ) Agarose gel images showing pEGFP-c1 plasmid DNA (150 ng) incubated with 0.1 μ M hAChE-T547 at different pH at 37 °C for 6 h. ( e ) Effects of Mg 2+ and (or) Ca 2+ on DNA cleavage activity of hAChE-T547. The hydrolysis buffer containing Mg 2+ and (or) Ca 2+ at the indicated concentration was pre-incubated in the presence or absence of ethylene glycol tetraacetic acid (EGTA)/EDTA at 37 °C for 1 h. hAChE-T547 and pEGFP-c1 plasmid DNA (150 ng) was then added and the incubation continued for a further 6 h. DNase I was used as a positive control. ( f ) The effects of G-actin on DNA cleavage activity of hAChE-T547. hAChE-T547 and DNase I were pre-incubated with G-actin at the indicated concentration in the hydrolysis buffer at 37 °C for 1 h. pEGFP-c1 plasmid DNA (150 ng) was then added and the incubation continued for a further 6 h. DNase I was used as a positive control. ( g ) Silver staining and western blot showing the purified human caspase-activated deoxyribonuclease (CAD) (Arg87–Lys323; predicted molecular mass: 31.9 kDa) (USCN Life Science, Wuhan, China). The anti-CAD antibody (Merck Millipore, Darmstadt, Germany, AB16926) was used at a dilution of 1:500. ( h ) Agarose gel images showing pEGFP-c1 plasmid DNA (150 ng) incubated with purified hAChE-T547 and CAD/DFF40, respectively, at the indicated concentration at 37 °C for 6 h. SC, supercoiled.
    Figure Legend Snippet: Purified synaptic acetylcholinesterase polypeptide cleaves DNA in a cell-free hydrolysis system. ( a ) Agarose gel images showing pEGFP-c1 plasmid DNA (150 ng) incubated with purified hAChE-T547 at the indicated concentration at 37 °C for 6 h. Eco RI-digested DNA (0.5 U/μl) was used to demarcate the linearized plasmid. Bovine serum albumin (BSA) was used as a negative control. ( b ) Agarose gel images of pEGFP-c1 plasmid DNA (150 ng) incubated with 0.5 μ M hAChE-T547 at 37 °C for the indicated time periods. ( c ) Agarose gel images of pEGFP-c1 plasmid DNA (150–600 ng) incubated with or without 0.5 μ M hAChE-T547 at 37 °C for 6 h. ( d ) Agarose gel images showing pEGFP-c1 plasmid DNA (150 ng) incubated with 0.1 μ M hAChE-T547 at different pH at 37 °C for 6 h. ( e ) Effects of Mg 2+ and (or) Ca 2+ on DNA cleavage activity of hAChE-T547. The hydrolysis buffer containing Mg 2+ and (or) Ca 2+ at the indicated concentration was pre-incubated in the presence or absence of ethylene glycol tetraacetic acid (EGTA)/EDTA at 37 °C for 1 h. hAChE-T547 and pEGFP-c1 plasmid DNA (150 ng) was then added and the incubation continued for a further 6 h. DNase I was used as a positive control. ( f ) The effects of G-actin on DNA cleavage activity of hAChE-T547. hAChE-T547 and DNase I were pre-incubated with G-actin at the indicated concentration in the hydrolysis buffer at 37 °C for 1 h. pEGFP-c1 plasmid DNA (150 ng) was then added and the incubation continued for a further 6 h. DNase I was used as a positive control. ( g ) Silver staining and western blot showing the purified human caspase-activated deoxyribonuclease (CAD) (Arg87–Lys323; predicted molecular mass: 31.9 kDa) (USCN Life Science, Wuhan, China). The anti-CAD antibody (Merck Millipore, Darmstadt, Germany, AB16926) was used at a dilution of 1:500. ( h ) Agarose gel images showing pEGFP-c1 plasmid DNA (150 ng) incubated with purified hAChE-T547 and CAD/DFF40, respectively, at the indicated concentration at 37 °C for 6 h. SC, supercoiled.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Incubation, Concentration Assay, Negative Control, Activity Assay, Positive Control, Silver Staining, Western Blot

    14) Product Images from "Molecular interactions of Escherichia coli ExoIX and identification of its associated 3?-5? exonuclease activity"

    Article Title: Molecular interactions of Escherichia coli ExoIX and identification of its associated 3?-5? exonuclease activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm396

    Protein interactions of ExoIX. ( A ) SDS–PAGE analysis of the protein–protein interactions of GST–Exo IX and lysate of  E. coli  MG1655. Cell free lysate of  E. coli  MG1655 was pre-incubated with either GST–ExoIX or GST for 1 h at 4°C. The mixtures were loaded onto a GSTrap column (GE Biosciences), washed and eluted with 10 mM glutathione. Of the proteins which specifically eluted with GST–Exo IX only one was visible in the final gel image (∼20 kDa). Lanes, MG, MG1655 cell free lysate; L, GSTrap load; FT, flow through; F6/7, eluted fractions. ( B ) SDS–PAGE analysis of pull-down assays with ExoIX-Sepharose. MG1655 lysate was treated with DNase I, prior to incubation with immobilized protein. Arrows indicate specific protein interactants which are present in the eluate from ExoIX-Sepharose (  Table 2 ) and absent in the BSA-Sepharose control. Abbreviations, L, MG1655 cell lysate; FT, flow through; BSA, BSA-Sepharose 4B eluate; IX, ExoIX-Sepharose eluate.
    Figure Legend Snippet: Protein interactions of ExoIX. ( A ) SDS–PAGE analysis of the protein–protein interactions of GST–Exo IX and lysate of E. coli MG1655. Cell free lysate of E. coli MG1655 was pre-incubated with either GST–ExoIX or GST for 1 h at 4°C. The mixtures were loaded onto a GSTrap column (GE Biosciences), washed and eluted with 10 mM glutathione. Of the proteins which specifically eluted with GST–Exo IX only one was visible in the final gel image (∼20 kDa). Lanes, MG, MG1655 cell free lysate; L, GSTrap load; FT, flow through; F6/7, eluted fractions. ( B ) SDS–PAGE analysis of pull-down assays with ExoIX-Sepharose. MG1655 lysate was treated with DNase I, prior to incubation with immobilized protein. Arrows indicate specific protein interactants which are present in the eluate from ExoIX-Sepharose ( Table 2 ) and absent in the BSA-Sepharose control. Abbreviations, L, MG1655 cell lysate; FT, flow through; BSA, BSA-Sepharose 4B eluate; IX, ExoIX-Sepharose eluate.

    Techniques Used: SDS Page, Incubation, Flow Cytometry

    15) Product Images from "Spo0A-Dependent Activation of an Extended -10 Region Promoter in Bacillus subtilis"

    Article Title: Spo0A-Dependent Activation of an Extended -10 Region Promoter in Bacillus subtilis

    Journal:

    doi: 10.1128/JB.188.4.1411-1418.2006

    DNase I protection assay analysis of the skf promoter with C-Spo0A. A 32 P-labeled nontemplate strand of skf promoter DNA was incubated with or without purified C-Spo0A (0 to 240 nM) and subjected to DNase I digestion for 1 min at 37°C (lanes a
    Figure Legend Snippet: DNase I protection assay analysis of the skf promoter with C-Spo0A. A 32 P-labeled nontemplate strand of skf promoter DNA was incubated with or without purified C-Spo0A (0 to 240 nM) and subjected to DNase I digestion for 1 min at 37°C (lanes a

    Techniques Used: Labeling, Incubation, Purification

    16) Product Images from "Extracellular Nucleases of Streptococcus equi subsp. zooepidemicus Degrade Neutrophil Extracellular Traps and Impair Macrophage Activity of the Host"

    Article Title: Extracellular Nucleases of Streptococcus equi subsp. zooepidemicus Degrade Neutrophil Extracellular Traps and Impair Macrophage Activity of the Host

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02468-16

    DNase activities of ENuc and 5Nuc. (A) ENuc and 5Nuc degrade calf thymus DNA. Banding indicates the remaining DNA, and lanes from left to right represent DNase I, reaction buffer, rENuc, and r5Nuc incubated with calf thymus DNA. (B) Wild-type  S. equi
    Figure Legend Snippet: DNase activities of ENuc and 5Nuc. (A) ENuc and 5Nuc degrade calf thymus DNA. Banding indicates the remaining DNA, and lanes from left to right represent DNase I, reaction buffer, rENuc, and r5Nuc incubated with calf thymus DNA. (B) Wild-type S. equi

    Techniques Used: Incubation

    17) Product Images from "Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair"

    Article Title: Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2005595

    Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.
    Figure Legend Snippet: Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.

    Techniques Used: Mutagenesis, High Throughput Screening Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Mouse Assay, CpG Methylation Assay, Non-Homologous End Joining

    18) Product Images from "Enhanced Orai1 and STIM1 expression as well as store operated Ca2+ entry in therapy resistant ovary carcinoma cells"

    Article Title: Enhanced Orai1 and STIM1 expression as well as store operated Ca2+ entry in therapy resistant ovary carcinoma cells

    Journal: Oncotarget

    doi:

    Effect of Akt inhibitor SH-6 on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of annexin V binding plotted against propidium iodide in therapy sensitive cells (sensitive) and therapy resistant (resistant) ovary carcinoma cells without (left panels) and with (right panels) a 24 h exposure to 10 μM SH-6 and without (control) and with (cisplatin) treatment with cisplatin (100 μM, 24 h). The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n = 6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or SH-6 (10 μM, black bars) prior to (control) and following treatment with cisplatin (100 μM, 24 h) (cisplatin). *** (p
    Figure Legend Snippet: Effect of Akt inhibitor SH-6 on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of annexin V binding plotted against propidium iodide in therapy sensitive cells (sensitive) and therapy resistant (resistant) ovary carcinoma cells without (left panels) and with (right panels) a 24 h exposure to 10 μM SH-6 and without (control) and with (cisplatin) treatment with cisplatin (100 μM, 24 h). The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n = 6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or SH-6 (10 μM, black bars) prior to (control) and following treatment with cisplatin (100 μM, 24 h) (cisplatin). *** (p

    Techniques Used: Binding Assay

    Effect of SOCE inhibitor 2-APB on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of a representative experiment of annexin V binding plotted against propidium iodide staining of therapy sensitive cells (sensitive) and therapy resistant ovary carcinoma cells (resistant) without (left panels) and with (right panels) a 24 h exposure to 50 μM 2-APB and with (cisplatin) and without (control) cisplatin (100 μM, 24 h) treatment. The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n =5-6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or 2-APB (50 μM, black bars) prior to (control) and following (cisplatin) treatment with cisplatin (100 μM, 24 h). *** (p
    Figure Legend Snippet: Effect of SOCE inhibitor 2-APB on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of a representative experiment of annexin V binding plotted against propidium iodide staining of therapy sensitive cells (sensitive) and therapy resistant ovary carcinoma cells (resistant) without (left panels) and with (right panels) a 24 h exposure to 50 μM 2-APB and with (cisplatin) and without (control) cisplatin (100 μM, 24 h) treatment. The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n =5-6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or 2-APB (50 μM, black bars) prior to (control) and following (cisplatin) treatment with cisplatin (100 μM, 24 h). *** (p

    Techniques Used: Binding Assay, Staining

    19) Product Images from "Enhanced Orai1 and STIM1 expression as well as store operated Ca2+ entry in therapy resistant ovary carcinoma cells"

    Article Title: Enhanced Orai1 and STIM1 expression as well as store operated Ca2+ entry in therapy resistant ovary carcinoma cells

    Journal: Oncotarget

    doi:

    Effect of Akt inhibitor SH-6 on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of annexin V binding plotted against propidium iodide in therapy sensitive cells (sensitive) and therapy resistant (resistant) ovary carcinoma cells without (left panels) and with (right panels) a 24 h exposure to 10 μM SH-6 and without (control) and with (cisplatin) treatment with cisplatin (100 μM, 24 h). The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n = 6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or SH-6 (10 μM, black bars) prior to (control) and following treatment with cisplatin (100 μM, 24 h) (cisplatin). *** (p
    Figure Legend Snippet: Effect of Akt inhibitor SH-6 on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of annexin V binding plotted against propidium iodide in therapy sensitive cells (sensitive) and therapy resistant (resistant) ovary carcinoma cells without (left panels) and with (right panels) a 24 h exposure to 10 μM SH-6 and without (control) and with (cisplatin) treatment with cisplatin (100 μM, 24 h). The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n = 6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or SH-6 (10 μM, black bars) prior to (control) and following treatment with cisplatin (100 μM, 24 h) (cisplatin). *** (p

    Techniques Used: Binding Assay

    Effect of SOCE inhibitor 2-APB on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of a representative experiment of annexin V binding plotted against propidium iodide staining of therapy sensitive cells (sensitive) and therapy resistant ovary carcinoma cells (resistant) without (left panels) and with (right panels) a 24 h exposure to 50 μM 2-APB and with (cisplatin) and without (control) cisplatin (100 μM, 24 h) treatment. The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n =5-6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or 2-APB (50 μM, black bars) prior to (control) and following (cisplatin) treatment with cisplatin (100 μM, 24 h). *** (p
    Figure Legend Snippet: Effect of SOCE inhibitor 2-APB on cisplatin induced apoptosis of therapy sensitive and therapy resistant ovary carcinoma cells A. Original dot plots of a representative experiment of annexin V binding plotted against propidium iodide staining of therapy sensitive cells (sensitive) and therapy resistant ovary carcinoma cells (resistant) without (left panels) and with (right panels) a 24 h exposure to 50 μM 2-APB and with (cisplatin) and without (control) cisplatin (100 μM, 24 h) treatment. The cells without loss of membrane integrity and externalized phosphatidylserine at the cell surface appear on the lower left quadrant of the dot plot. B. Arithmetic means (± SEM, n =5-6) of the percentage of therapy sensitive (sensitive) and therapy resistant (resistant) ovary carcinoma cells binding Annexin V following 24 h exposure to DMSO (1‰, white bars) or 2-APB (50 μM, black bars) prior to (control) and following (cisplatin) treatment with cisplatin (100 μM, 24 h). *** (p

    Techniques Used: Binding Assay, Staining

    20) Product Images from "AphB Influences Acid Tolerance of Vibrio vulnificus by Activating Expression of the Positive Regulator CadC"

    Article Title: AphB Influences Acid Tolerance of Vibrio vulnificus by Activating Expression of the Positive Regulator CadC

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00533-06

    Identification of AphB binding site using DNase I protection analysis (A) and sequence analysis of the cadC upstream region (B). (A) The 32 P-labeled 298-bp cadC regulatory region was incubated with increasing amounts of AphB and then digested with DNase
    Figure Legend Snippet: Identification of AphB binding site using DNase I protection analysis (A) and sequence analysis of the cadC upstream region (B). (A) The 32 P-labeled 298-bp cadC regulatory region was incubated with increasing amounts of AphB and then digested with DNase

    Techniques Used: Binding Assay, Sequencing, Labeling, Incubation

    21) Product Images from "Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces"

    Article Title: Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces

    Journal: Nanoscale Research Letters

    doi: 10.1007/s11671-009-9350-6

    AFM images of DNA reaction of digestion by DNase I. Height scales = 8 nm except for ( a ). a DNA topography before digestion. Height scale = 2 nm. b DNA fragments just after a DPN process. c DNA fragments after DPN 0.5 h. d Traces of DNA after DPN 10 h
    Figure Legend Snippet: AFM images of DNA reaction of digestion by DNase I. Height scales = 8 nm except for ( a ). a DNA topography before digestion. Height scale = 2 nm. b DNA fragments just after a DPN process. c DNA fragments after DPN 0.5 h. d Traces of DNA after DPN 10 h

    Techniques Used:

    22) Product Images from "Binding Site Recognition by Rns, a Virulence Regulator in the AraC Family"

    Article Title: Binding Site Recognition by Rns, a Virulence Regulator in the AraC Family

    Journal: Journal of Bacteriology

    doi:

    Three-dimensional representation of Rns binding sites I and II. The positions within each binding site that remain accessible or become hypersensitive to DNase I cleavage upon MBP::Rns binding are indicated by diamonds. Within each binding site the three thymine C5-methyl groups that MBP::Rns has hydrophobic interactions with are shown by solid circles. To align these thymines so that they appear in the same orientation in the figure, the sequence of binding site I has been inverted. The numbering is relative to the transcription start site of P coo .
    Figure Legend Snippet: Three-dimensional representation of Rns binding sites I and II. The positions within each binding site that remain accessible or become hypersensitive to DNase I cleavage upon MBP::Rns binding are indicated by diamonds. Within each binding site the three thymine C5-methyl groups that MBP::Rns has hydrophobic interactions with are shown by solid circles. To align these thymines so that they appear in the same orientation in the figure, the sequence of binding site I has been inverted. The numbering is relative to the transcription start site of P coo .

    Techniques Used: Binding Assay, Sequencing

    DNase I footprints of MBP::Rns bound to P coo . The vertical bars indicate the positions of Rns binding sites I and II. The open rectangles show the positions of the promoter −10 and −35 hexamers. The numbering is relative to the transcription start site. (A) MBP::Rns bound to the coding strand of P coo DNA. Lanes 1 and 8 are without MBP::Rns; lanes 2 through 7 contain 300, 200, 133, 89, 59, and 40 nM MBP::Rns. (B) MBP::Rns bound to the noncoding strand of P coo DNA. Lanes 1 and 6 are without MBP::Rns; lanes 2 through 5 contain 200, 133, 89, and 60 nM MBP::Rns. The lanes labeled GA and TC contain Maxam-Gilbert sequence ladders.
    Figure Legend Snippet: DNase I footprints of MBP::Rns bound to P coo . The vertical bars indicate the positions of Rns binding sites I and II. The open rectangles show the positions of the promoter −10 and −35 hexamers. The numbering is relative to the transcription start site. (A) MBP::Rns bound to the coding strand of P coo DNA. Lanes 1 and 8 are without MBP::Rns; lanes 2 through 7 contain 300, 200, 133, 89, 59, and 40 nM MBP::Rns. (B) MBP::Rns bound to the noncoding strand of P coo DNA. Lanes 1 and 6 are without MBP::Rns; lanes 2 through 5 contain 200, 133, 89, and 60 nM MBP::Rns. The lanes labeled GA and TC contain Maxam-Gilbert sequence ladders.

    Techniques Used: Binding Assay, Labeling, Sequencing

    Summary of DNase I protection and uracil interference assays. The nucleotides within P coo that were protected from DNase I by MBP::Rns binding are shaded. Uracil substitutions that interfere with MBP::Rns binding are indicated by the letter U. The transcription start site is indicated by an arrow.
    Figure Legend Snippet: Summary of DNase I protection and uracil interference assays. The nucleotides within P coo that were protected from DNase I by MBP::Rns binding are shaded. Uracil substitutions that interfere with MBP::Rns binding are indicated by the letter U. The transcription start site is indicated by an arrow.

    Techniques Used: Binding Assay

    23) Product Images from "Reconstitution of Human ?-Globin Locus Control Region Hypersensitive Sites in the Absence of Chromatin Assembly"

    Article Title: Reconstitution of Human ?-Globin Locus Control Region Hypersensitive Sites in the Absence of Chromatin Assembly

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.8.2629-2640.2001

    Reconstitution of DNase I HS sites in chromatin-assembled LCR templates. (A) Outline of the general strategy for immobilizing the LCR on magnetic beads (step 1), the chromatin assembly reaction (step 2), and the incubation with erythroid and non-erythroid cell extracts (step 3). (B) MNase digestion pattern of chromatin-assembled pRS/LCR. pRS/LCR was attached to the beads and incubated with Drosophila chromatin assembly extract (Materials and Methods). The beads were washed on a magnet and digested with MNase for 15, 30, or 60 s, after which the DNA was isolated and separated in a 1.3% agarose gel. The DNA was then blotted to a nylon membrane and hybridized to a 32 P-labeled fragment corresponding to the 3′ region of HS2. (C) DNase I HS site mapping of unassembled and chromatin-assembled pRS/LCR. For analyzing DNase I sensitivity in the unassembled construct, pRS/LCR was incubated in buffer A (No protein) or in buffer A with 100 μg of MEL protein extract (MEL) for 45 min at 30°C and then digested with increasing concentrations of DNase I. For analyzing DNase I sensitivity in chromatin-assembled templates, pRS/LCR was first assembled into chromatin as described for panel B and then incubated in buffer A containing no protein, 100 μg of HeLa protein extract (HeLa), or 100 μg of MEL protein extract (MEL) for 45 min at 30°C. The isolated DNA was then digested with Eco RI, processed, and analyzed as described for panel B. (D) DNase I hypersensitivity mapping on chromatin-assembled pRS/LCR preincubated with MEL protein extracts. pRS/LCR was incubated with 100 μg of protein extract from MEL cells (MEL) for 45 min at 30°C prior to chromatin assembly and then digested with increasing concentrations of DNase I. The samples were processed and analyzed as described for panel C. In lanes HS2, HS3, and HS3.1, restriction fragments marking the positions of the HS2 and HS3 core enhancers were included (HS2, Eco RI/ Hin dIII, marking the 5′ end of HS2; HS3, Eco RI/ Spe I, marking the 5′ end of HS3; HS3.1, Eco RI/ Sca I, corresponding to the 3′ end of HS3).
    Figure Legend Snippet: Reconstitution of DNase I HS sites in chromatin-assembled LCR templates. (A) Outline of the general strategy for immobilizing the LCR on magnetic beads (step 1), the chromatin assembly reaction (step 2), and the incubation with erythroid and non-erythroid cell extracts (step 3). (B) MNase digestion pattern of chromatin-assembled pRS/LCR. pRS/LCR was attached to the beads and incubated with Drosophila chromatin assembly extract (Materials and Methods). The beads were washed on a magnet and digested with MNase for 15, 30, or 60 s, after which the DNA was isolated and separated in a 1.3% agarose gel. The DNA was then blotted to a nylon membrane and hybridized to a 32 P-labeled fragment corresponding to the 3′ region of HS2. (C) DNase I HS site mapping of unassembled and chromatin-assembled pRS/LCR. For analyzing DNase I sensitivity in the unassembled construct, pRS/LCR was incubated in buffer A (No protein) or in buffer A with 100 μg of MEL protein extract (MEL) for 45 min at 30°C and then digested with increasing concentrations of DNase I. For analyzing DNase I sensitivity in chromatin-assembled templates, pRS/LCR was first assembled into chromatin as described for panel B and then incubated in buffer A containing no protein, 100 μg of HeLa protein extract (HeLa), or 100 μg of MEL protein extract (MEL) for 45 min at 30°C. The isolated DNA was then digested with Eco RI, processed, and analyzed as described for panel B. (D) DNase I hypersensitivity mapping on chromatin-assembled pRS/LCR preincubated with MEL protein extracts. pRS/LCR was incubated with 100 μg of protein extract from MEL cells (MEL) for 45 min at 30°C prior to chromatin assembly and then digested with increasing concentrations of DNase I. The samples were processed and analyzed as described for panel C. In lanes HS2, HS3, and HS3.1, restriction fragments marking the positions of the HS2 and HS3 core enhancers were included (HS2, Eco RI/ Hin dIII, marking the 5′ end of HS2; HS3, Eco RI/ Spe I, marking the 5′ end of HS3; HS3.1, Eco RI/ Sca I, corresponding to the 3′ end of HS3).

    Techniques Used: Magnetic Beads, Incubation, Isolation, Agarose Gel Electrophoresis, Labeling, Construct

    Erythroid cell-specific generation of LCR DNase I and S1 nuclease HS sites in the absence of chromatin assembly. (A) Representation of the human β-globin LCR indicating the positions of Eco RI sites that were used to map hypersensitivity in HS2 and HS3. (B) The LCR (600 ng) was attached to magnetic beads and incubated for 45 min at 30°C with no protein, with 100 μg of HeLa extract, or with 100 μg of MEL protein extract, as indicated. Subsequently, aliquots were digested with increasing concentrations of either DNase I or S1 nuclease, as indicated. The DNA was digested with Eco RI, subjected to electrophoresis, blotted to a nylon membrane, and then hybridized to a 32 P-labeled probe corresponding to the 3′ region of HS2.
    Figure Legend Snippet: Erythroid cell-specific generation of LCR DNase I and S1 nuclease HS sites in the absence of chromatin assembly. (A) Representation of the human β-globin LCR indicating the positions of Eco RI sites that were used to map hypersensitivity in HS2 and HS3. (B) The LCR (600 ng) was attached to magnetic beads and incubated for 45 min at 30°C with no protein, with 100 μg of HeLa extract, or with 100 μg of MEL protein extract, as indicated. Subsequently, aliquots were digested with increasing concentrations of either DNase I or S1 nuclease, as indicated. The DNA was digested with Eco RI, subjected to electrophoresis, blotted to a nylon membrane, and then hybridized to a 32 P-labeled probe corresponding to the 3′ region of HS2.

    Techniques Used: Magnetic Beads, Incubation, Electrophoresis, Labeling

    24) Product Images from "Intercellular adhesion and biocide resistance in nontypeable Haemophilus influenzae biofilms"

    Article Title: Intercellular adhesion and biocide resistance in nontypeable Haemophilus influenzae biofilms

    Journal: Microbial pathogenesis

    doi: 10.1016/j.micpath.2009.01.004

    DNase I and proteinase K sensitize NTHi NJ9725 biofilms to killing by CPC. (A) Biofilms cultured for 24 in microtiter plates were rinsed with water and treated for 10 min with DNase I buffer (mock pre-treatment) or DNase I buffer containing 1 mg mL -1
    Figure Legend Snippet: DNase I and proteinase K sensitize NTHi NJ9725 biofilms to killing by CPC. (A) Biofilms cultured for 24 in microtiter plates were rinsed with water and treated for 10 min with DNase I buffer (mock pre-treatment) or DNase I buffer containing 1 mg mL -1

    Techniques Used: Cell Culture

    DNase I and proteinase K inhibit NTHi biofilm formation. (A) NTHi strain NJ9725 was cultured in polystyrene tubes in BHI broth or BHI broth supplemented with 1 mg mL -1 of DNase I or proteinase K. After increasing amounts of time, tubes were washed with
    Figure Legend Snippet: DNase I and proteinase K inhibit NTHi biofilm formation. (A) NTHi strain NJ9725 was cultured in polystyrene tubes in BHI broth or BHI broth supplemented with 1 mg mL -1 of DNase I or proteinase K. After increasing amounts of time, tubes were washed with

    Techniques Used: Cell Culture

    Detachment of 24-h-old NTHi biofilms by DNase I and proteinase K. All enzymes were tested in enzyme buffer at a concentration of 1 mg mL -1 . (A) Detachment of biofilms in 1 h by proteinase K. (B) Time course for proteinase K-induced detachment of NJ9725
    Figure Legend Snippet: Detachment of 24-h-old NTHi biofilms by DNase I and proteinase K. All enzymes were tested in enzyme buffer at a concentration of 1 mg mL -1 . (A) Detachment of biofilms in 1 h by proteinase K. (B) Time course for proteinase K-induced detachment of NJ9725

    Techniques Used: Concentration Assay

    Fluid convection through 24-h-old NTHi NJ9725 biofilms cultured in centrifugal filter devices. Biofilms were cultured in BHI broth or BHI broth supplemented with 1 mg mL -1 DNase I or proteinase K. The devices were subjected to low-speed centrifugation
    Figure Legend Snippet: Fluid convection through 24-h-old NTHi NJ9725 biofilms cultured in centrifugal filter devices. Biofilms were cultured in BHI broth or BHI broth supplemented with 1 mg mL -1 DNase I or proteinase K. The devices were subjected to low-speed centrifugation

    Techniques Used: Convection, Cell Culture, Centrifugation

    25) Product Images from "SOS Regulation of the Type III Secretion System of Enteropathogenic Escherichia coli ▿"

    Article Title: SOS Regulation of the Type III Secretion System of Enteropathogenic Escherichia coli ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01859-06

    DNase I protection assays to establish purified LexA protein binding in LEE and recA regulatory DNA fragments in vitro. (A) The region of protection from DNase I cleavage, demonstrating LexA binding at the LEE2 / LEE3 promoters, is indicated by a vertical
    Figure Legend Snippet: DNase I protection assays to establish purified LexA protein binding in LEE and recA regulatory DNA fragments in vitro. (A) The region of protection from DNase I cleavage, demonstrating LexA binding at the LEE2 / LEE3 promoters, is indicated by a vertical

    Techniques Used: Purification, Protein Binding, In Vitro, Binding Assay

    26) Product Images from "High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers"

    Article Title: High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp437

    Comparison of PNA oligomer and TFO binding to the complementary dsDNA target at pH 6.3. ( A ) Autoradiograph showing DNase I footprint formed by PNA1846 (lanes 3–9) and TFO1 (lanes 11–17) bound to the complementary dsDNA target from p322. The position of the sequence target is indicated adjacent to the A/G sequence reaction (lane 1). Asterisk indicates the position of the 32 P-label. Lanes 2 and 10 are controls without oligomer. ( B ) Quantitative analysis of the data shown in (A). Percentage DNase I protection as a function of PNA1846 or TFO1 concentration. ( C ) Autoradiograph showing PNA1846 binding to the complementary sequence target in p322 as analysed by gel-shift analysis. The following PNA1846 concentrations (µM) were used: w/o (lane 1), 0.002 (lane 2), 0.006 (lane 3), 0.02 (lane 4), 0.06 (lane 5), 0.17 (lane 6), 0.5 (lane 7), 1.5 (lane 8) and 4.5 (lane 9). ( D ) Percentage PD-complex (triplex or invasion) formed as a function of PNA concentration is shown. Free dsDNA is included for reference (indicated as duplex). Error bars indicate standard error of the mean (SEM) of six experimental repetitions. Notice the bell-shaped curve for triplex formation. Also notice the logarithmic scale of the x -axis.
    Figure Legend Snippet: Comparison of PNA oligomer and TFO binding to the complementary dsDNA target at pH 6.3. ( A ) Autoradiograph showing DNase I footprint formed by PNA1846 (lanes 3–9) and TFO1 (lanes 11–17) bound to the complementary dsDNA target from p322. The position of the sequence target is indicated adjacent to the A/G sequence reaction (lane 1). Asterisk indicates the position of the 32 P-label. Lanes 2 and 10 are controls without oligomer. ( B ) Quantitative analysis of the data shown in (A). Percentage DNase I protection as a function of PNA1846 or TFO1 concentration. ( C ) Autoradiograph showing PNA1846 binding to the complementary sequence target in p322 as analysed by gel-shift analysis. The following PNA1846 concentrations (µM) were used: w/o (lane 1), 0.002 (lane 2), 0.006 (lane 3), 0.02 (lane 4), 0.06 (lane 5), 0.17 (lane 6), 0.5 (lane 7), 1.5 (lane 8) and 4.5 (lane 9). ( D ) Percentage PD-complex (triplex or invasion) formed as a function of PNA concentration is shown. Free dsDNA is included for reference (indicated as duplex). Error bars indicate standard error of the mean (SEM) of six experimental repetitions. Notice the bell-shaped curve for triplex formation. Also notice the logarithmic scale of the x -axis.

    Techniques Used: Binding Assay, Autoradiography, Sequencing, Concentration Assay, Electrophoretic Mobility Shift Assay

    27) Product Images from "A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence"

    Article Title: A novel extracellular vesicle-associated endodeoxyribonuclease helps Streptococcus pneumoniae evade neutrophil extracellular traps and is required for full virulence

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25865-z

    Pneumococcal secretome possesses DNase activity. ( a ) Calf thymus DNA (CT DNA) was incubated in the presence or absence of secretome from lytA deficient mutant of S. pneumoniae strain D39 grown in THY medium (D39Δ lytA sec). The samples were incubated at 37 °C for 30 min. The processed samples were resolved on an ethidium bromide stained agarose gel (0.8%; uncropped image). Bovine pancreatic DNase I (BP DNase) was used as a positive control. Secretome only and THY medium was taken as negative and vehicle control, respectively. ( b ) DNase activity of the secretome (sec) derived from pneumococcal strains D39 (1; served as the positive control), TIGR4 (2), ATCC 6301 (3), ATCC 6314 (4) and WU2 (5). The DNase activity observed with the secretome obtained from D39Δ lytA propagated in chemically defined medium (CDM) is heat labile ( c ), and sensitive to treatment with proteinase K ( d ) and EDTA ( e ). Linear double-stranded plasmid DNA was used as the substrate in panels c-e. The molecular mass marker (in Kb) is shown. Heat, proteinase K and EDTA treatment is indicated by ‘HT’, ‘PK’ and ‘EDTA’, respectively. ( f ) Secretome from D39Δ lytA was resolved on SDS-PAG bearing calf thymus DNA (30 μg/ml) followed by ethidium bromide staining as described in the Materials and Methods . Three dark bands corresponding to DNA degradation were observed against fluorescent background (indicated using arrowheads). A portion of the gel has been enlarged to highlight the three bands. ( g .
    Figure Legend Snippet: Pneumococcal secretome possesses DNase activity. ( a ) Calf thymus DNA (CT DNA) was incubated in the presence or absence of secretome from lytA deficient mutant of S. pneumoniae strain D39 grown in THY medium (D39Δ lytA sec). The samples were incubated at 37 °C for 30 min. The processed samples were resolved on an ethidium bromide stained agarose gel (0.8%; uncropped image). Bovine pancreatic DNase I (BP DNase) was used as a positive control. Secretome only and THY medium was taken as negative and vehicle control, respectively. ( b ) DNase activity of the secretome (sec) derived from pneumococcal strains D39 (1; served as the positive control), TIGR4 (2), ATCC 6301 (3), ATCC 6314 (4) and WU2 (5). The DNase activity observed with the secretome obtained from D39Δ lytA propagated in chemically defined medium (CDM) is heat labile ( c ), and sensitive to treatment with proteinase K ( d ) and EDTA ( e ). Linear double-stranded plasmid DNA was used as the substrate in panels c-e. The molecular mass marker (in Kb) is shown. Heat, proteinase K and EDTA treatment is indicated by ‘HT’, ‘PK’ and ‘EDTA’, respectively. ( f ) Secretome from D39Δ lytA was resolved on SDS-PAG bearing calf thymus DNA (30 μg/ml) followed by ethidium bromide staining as described in the Materials and Methods . Three dark bands corresponding to DNA degradation were observed against fluorescent background (indicated using arrowheads). A portion of the gel has been enlarged to highlight the three bands. ( g .

    Techniques Used: Activity Assay, Incubation, Mutagenesis, Size-exclusion Chromatography, Staining, Agarose Gel Electrophoresis, Positive Control, Derivative Assay, Plasmid Preparation, Marker

    Degradation of NETs by pneumococcal secretome, EVs and rTatD. Human neutrophils were treated with 25 nM PMA for 2 h to release NETs (stimulated). NETs were incubated with ( a ) secretome from D39 or D39Δ tatD (10 μg/ml). Bovine pancreatic DNase I (BP DNase) was used as the positive control. NETs were visualized using a confocal microscope following staining with DAPI (blue) and anti-myeloperoxidase antibody (MPO, green). ( b ) NETs incubated with EVs from D39 or D39Δ tatD . ( c ) NETs treated with rTatD. A non-relevant pneumococcal recombinant protein (rSP_0149) was used as a negative control. Scale bars represent 20 micron. Arrowheads are used to indicate NETs. ( d – f ) Twenty microscopic fields were selected at random for quantitative analysis using ImageJ software. NET degradation is represented as the fraction of NETs present in the treated sample as compared to PMA stimulated sample (control) expressed in percentage terms. Two tailed unpaired t test was used for statistical analysis. Error bars represent mean ± sem. Each experiment was performed twice and data from a representative experiment is shown. All the images shown in panels a-c are uncropped.
    Figure Legend Snippet: Degradation of NETs by pneumococcal secretome, EVs and rTatD. Human neutrophils were treated with 25 nM PMA for 2 h to release NETs (stimulated). NETs were incubated with ( a ) secretome from D39 or D39Δ tatD (10 μg/ml). Bovine pancreatic DNase I (BP DNase) was used as the positive control. NETs were visualized using a confocal microscope following staining with DAPI (blue) and anti-myeloperoxidase antibody (MPO, green). ( b ) NETs incubated with EVs from D39 or D39Δ tatD . ( c ) NETs treated with rTatD. A non-relevant pneumococcal recombinant protein (rSP_0149) was used as a negative control. Scale bars represent 20 micron. Arrowheads are used to indicate NETs. ( d – f ) Twenty microscopic fields were selected at random for quantitative analysis using ImageJ software. NET degradation is represented as the fraction of NETs present in the treated sample as compared to PMA stimulated sample (control) expressed in percentage terms. Two tailed unpaired t test was used for statistical analysis. Error bars represent mean ± sem. Each experiment was performed twice and data from a representative experiment is shown. All the images shown in panels a-c are uncropped.

    Techniques Used: Incubation, Positive Control, Microscopy, Staining, Recombinant, Negative Control, Software, Two Tailed Test

    28) Product Images from "Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence"

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074662

    RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).
    Figure Legend Snippet: RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).

    Techniques Used: Stable Transfection, Expressing, Staining, Transfection, Immunoprecipitation, Western Blot

    Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.
    Figure Legend Snippet: Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.

    Techniques Used: Stable Transfection, Expressing, Sequencing, Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Staining, Transfection, Western Blot, Marker, Mutagenesis, Immunoprecipitation

    Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).
    Figure Legend Snippet: Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).

    Techniques Used: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation

    Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.
    Figure Legend Snippet: Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.

    Techniques Used: Fluorescence, Förster Resonance Energy Transfer, Stable Transfection, Transduction, Transfection, Expressing, Software, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation

    Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).
    Figure Legend Snippet: Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).

    Techniques Used: Infection, Transduction, Expressing, Staining, Stable Transfection, Immunoprecipitation, Western Blot

    29) Product Images from "An epiblast stem cell-derived multipotent progenitor population for axial extension"

    Article Title: An epiblast stem cell-derived multipotent progenitor population for axial extension

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.168187

    Comparison between in vitro protocols to produce NMP-like cells. ) and did not split/passage the cells, which were grown for 5 days in the same flask in the neural or mesodermal conditions. We named the resulting populations ES-neuro/ES-neuroF and ES-meso/ES-mesoF for those with an ES-NMP/ES-NMPF origin, and Epi-neuro and Epi-meso for those with an Epi-NMP origin. (C) Confocal immunofluorescent images of EpiSCs, and ES-NMP, ES-NMPF and Epi-NMP cultures on their 3rd day, and an Epi-meso culture on its 2nd day. Hoechst (nuclei) is in grey, Oct4 in red, Sox2 in green and T in magenta. The composite image of Sox2 (green) and T (magenta) is presented on the right-hand side. (D) Quantification plots of the fluorescence intensity in arbitrary units (a.u.) representing the protein levels in a cell. Each point represents a cell. The x -axis and y -axis represent the fluorescence intensity of either Sox2, T and Oct4. The numbers next to the T versus Sox2 plot indicate the percentage of cells that co-express Sox2 and T under the different conditions. The mean and the coefficient of variation (CV) of the distribution of Sox2, T and Oct4 across the different conditions are presented on the right-hand side: EpiSCs in orange, ES-NMP in yellow, ES-NMPF in turquoise, Epi-NMP in purple and Epi-meso in green.
    Figure Legend Snippet: Comparison between in vitro protocols to produce NMP-like cells. ) and did not split/passage the cells, which were grown for 5 days in the same flask in the neural or mesodermal conditions. We named the resulting populations ES-neuro/ES-neuroF and ES-meso/ES-mesoF for those with an ES-NMP/ES-NMPF origin, and Epi-neuro and Epi-meso for those with an Epi-NMP origin. (C) Confocal immunofluorescent images of EpiSCs, and ES-NMP, ES-NMPF and Epi-NMP cultures on their 3rd day, and an Epi-meso culture on its 2nd day. Hoechst (nuclei) is in grey, Oct4 in red, Sox2 in green and T in magenta. The composite image of Sox2 (green) and T (magenta) is presented on the right-hand side. (D) Quantification plots of the fluorescence intensity in arbitrary units (a.u.) representing the protein levels in a cell. Each point represents a cell. The x -axis and y -axis represent the fluorescence intensity of either Sox2, T and Oct4. The numbers next to the T versus Sox2 plot indicate the percentage of cells that co-express Sox2 and T under the different conditions. The mean and the coefficient of variation (CV) of the distribution of Sox2, T and Oct4 across the different conditions are presented on the right-hand side: EpiSCs in orange, ES-NMP in yellow, ES-NMPF in turquoise, Epi-NMP in purple and Epi-meso in green.

    Techniques Used: In Vitro, Fluorescence

    30) Product Images from "The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells"

    Article Title: The transcription factor Zfp90 regulates the self-renewal and differentiation of hematopoietic stem cells

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-018-0721-8

    Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p
    Figure Legend Snippet: Zfp90 cooperates with the NURF complex to regulate Hoxa9 expression. a Relative expression of representative HSC-proliferation-related genes. HSCs were isolated from Zfp90 +/+ and Zfp90 −/− mice 16 weeks after transplantation. b – d Analysis of representative gene expression in Bptf -, Snf2l - or Rbbp4 -deleted HSCs and WT control. Two sgRNAs were used for the deletion of Bptf, Snf2l or Rbbp4. e Analysis of Zfp90 enrichment on Hoxa9 promoter in LSK (Lin − Sca-1 + c-Kit + ) cells via ChIP assays. f Analysis of the direct interaction of Zfp90 with Hoxa9 promoter via EMSA assays. g Myc-Zfp90, pTK, and pGL3- Hoxa9 WT (region: −2000 to 0) or Mutant (Mut region: deletion of −550 to −800) promoter were transfected into 293T cells for luciferase assay. h , i Analysis of Bptf or Snf2l enrichment on Hoxa9 promoter in Zfp90 +/+ or Zfp90 −/− LSKs. j Accessibility of Hoxa9 promoter to DNase I in Zfp90 +/+ or Zfp90 −/− LSKs was assessed. * p

    Techniques Used: Expressing, Isolation, Mouse Assay, Transplantation Assay, Chromatin Immunoprecipitation, Mutagenesis, Transfection, Luciferase

    31) Product Images from "Interaction of Ler at the LEE5 (tir) Operon of Enteropathogenic Escherichia coli"

    Article Title: Interaction of Ler at the LEE5 (tir) Operon of Enteropathogenic Escherichia coli

    Journal: Infection and Immunity

    doi: 10.1128/IAI.71.1.384-392.2003

    DNase I footprint of Ler protein on the upstream LEE5 regulatory region. Concentrations of Ler protein (in micrograms per milliliter) are indicated at the top. (A) DNase I protection of the LEE5 coding strand. The Ler protein protected a region of DNA from DNase I digestion from positions −190 to −73, which is represented by an open bar. A DNase I-hypersensitive site was observed at position −111 on the coding strand and is indicated by an arrow. (B) DNase I protection of the LEE5 noncoding strand. Ler protein protected a region of DNA from positions −190 to −79, which is represented by an open bar. At higher concentrations of Ler protein, the regions of protection extended to positions −60 and −221 on the coding and noncoding strands, respectively, as indicated by shaded bars.
    Figure Legend Snippet: DNase I footprint of Ler protein on the upstream LEE5 regulatory region. Concentrations of Ler protein (in micrograms per milliliter) are indicated at the top. (A) DNase I protection of the LEE5 coding strand. The Ler protein protected a region of DNA from DNase I digestion from positions −190 to −73, which is represented by an open bar. A DNase I-hypersensitive site was observed at position −111 on the coding strand and is indicated by an arrow. (B) DNase I protection of the LEE5 noncoding strand. Ler protein protected a region of DNA from positions −190 to −79, which is represented by an open bar. At higher concentrations of Ler protein, the regions of protection extended to positions −60 and −221 on the coding and noncoding strands, respectively, as indicated by shaded bars.

    Techniques Used:

    32) Product Images from "Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence"

    Article Title: Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074662

    RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).
    Figure Legend Snippet: RFP-LANA associated with GFP-fused histone H1 and H2B. A. Chromosome spreads of 293T cells stably expressing GFP-H1 and GFP-H2B with RFP-LANA. GFP-H1 and GFP-H2B uniformly stained the entire chromosome and RFP-LANA showed distinct punctate localization on the chromosome detected by DAPI staining. B. 293T cells transfected with RFP-LANA and NLS-myc (lane 1), GFP-H1myc (lane 2) and GFP-H2Bmyc (lane 3) were harvested after 48 h post-transfection and lysed in RIPA buffer for immunoprecipitation with anti-myc antibody. Lysates from the above-mentioned transfection were treated with DNase I in second set before anti-myc immunoprecipitation. Bright band of RFP-LANA was detected in Myc-IP panels with GFP-H2Bmyc in untreated (lane 6) as well as DNase I treated panels (lane 12). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the input and IP lanes were detected with anti-myc WB and are indicated with red triangle. C . 10% of the above-transfected cells were passaged and allowed to grow for 96 h before lysing them for anti-myc immunoprecipitation. Lysates were either untreated or DNase I treated before anti-myc immuneprecipitation. Co-precipitating RFP-LANA was detected using anti-LANA western blot (IB:LANA). GFP-NLS-myc, GFP-H1myc and GFP-H2Bmyc in the inputs and IP lanes were detected with anti-myc WB (IB:myc).

    Techniques Used: Stable Transfection, Expressing, Staining, Transfection, Immunoprecipitation, Western Blot

    Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.
    Figure Legend Snippet: Stably expressing LANA 1–32 aa polypeptide bond to histone H2B. A. Schematic showing the strategy for generating GFP-NLS myc. Oligo containing a Nuclear Localization Sequence (NLS) of EBNA1 with two-tandem myc tag epitope was cloned at BamHI and XbaI sites (MCS) of pEFGCP-C1 vector to generate GFP fused with NLS and myc tag (GFP-NLS-myc). LANA 1–32 aa was PCR amplified with primers flanked with EcoRI and BamHI sites on the 5′ and 3′ respectively. GFP-NLS-myc was digested with EcoRI and BamHI, which released EBNA1 NLS, to clone LANA 1–32 aa (GFP-1–32 aa-myc). B. BJAB stably expressing GFP-NLS-myc or GFP-LANA 1–32myc was subjected for chromosome spreads and the nuclei were stained with propidium iodide (PI). GFP-LANA1–32myc completely painted the chromosomes whereas GFP-NLS-myc localized to nucleus but did not stain the chromosome. C. LANA 1–32 aa sequence showing CBD (5–15 aa) and its alanine substitution mutants highlighted in yellow. D. Histone H1 tagged with HA were transfected with GFP-NLS-myc (control) (lane 1) and GFP-LANA1–32 aa with wt CBD (lane 2) and its alanine substitution mutants (lanes 3–7 corresponding to mutants 3–7 in panel C. WB blot with anti-HA antibody showed a band of H1 with wt CBD LANA indicated with red triangle in the myc IP panel. Input showed uniformed expression of histone H1 in the input lanes. GFP-NLS-myc or GFP-LANA 1–32 and its mutants were detected with anti-myc in the input as well as IP panels. M shows the protein marker lane. Non-specific signals were detected below the red triangle in HA:WB panel. E. HA tagged histone H1 and H2B were co-transfected with GFP-NLS-myc (lane 1) or GFP-LANA1–32 wt (lane 2) and CBD mutants (lanes 3–7 corresponding to the mutants in panel C). Precipitation of GFP-NLS-myc and LANA 1–32 and it mutants showed co-precipitation of H2B (indicated with red triangle) with wt CBD containing LANA 1–32 (lane 2) and relatively lower amount with mutant 14-TG-15 (lane 6). GFP-NLS-myc and GFP-LANA1–32 and its mutants were detected with anti-myc blot in input as well as myc:IP lanes. IgG light chain was detected in HA:WB panel. F. Cells co-transfected with H1-HA and H2B-HA and GFP-NLS-myc or GFP-LANA1–32 aa and its mutants were lysed and the lysates were treated with 50 ug of DNase I for 45 min before immunoprecipitation. Co-precipitating H2B was detected in LANA 1–32 aa with wt CBD (lane 2). Both histones expressed in all the lanes detected by anti-HA WB. GFP-NLS-myc and LANA1–32 along with its mutants were detected with anti-myc WB. IgG light chain was detected in HA:WB panel above the H2B specific band.

    Techniques Used: Stable Transfection, Expressing, Sequencing, Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Staining, Transfection, Western Blot, Marker, Mutagenesis, Immunoprecipitation

    Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).
    Figure Legend Snippet: Immunoprecipitation of endogenous histones with LANA-N and LANA-FL. A. 293T cells were transfected with GFP-NLS-myc (control) or GFP-LANA-N (1–340 aa)-myc or its alanine substitution mutants (5–7 aa to alanine) and other respective mutants. Lysates were subjected to anti-myc IP without any treatment and immunoprecipitating GFP fusion proteins were detected with anti-myc antibody (Myc:WB panel, red triangle). Endogenous levels of histones were detected using anti-histone H1 (H1:WB) and anti-H2B (H2B:WB) antibodies (indicated red triangle). IgG light chain was detected in H1:WB panel in all the lanes. B. Lysate from 293T cells transfected with GFP-NLS-myc (control) or GFP-LANA-N and its alanine mutants were treated with 45 ug DNase I for 45 min before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected with specific antibodies (red triangle). IgG light chain was detected in H1:WB panel in all the lanes. C. Schematic of LANA-FL with marked CBD (5–15 aa). Mutants 2–6 were alanine substitution mutants of CBD. D. 293 T cells were transfected with myc vector (lane Vec) or myc tagged LANA-FL (lane 1) and its alanine substitution mutants (lanes 2–6). Cell lysate from the transfected cells were subjected for immunoprecipitation with anti-myc antibody followed detection of LANA and its mutants in anti-myc WB (WB:myc). Histone H1 and histone H2B were detected with specific antibodies (red triangle). E. Cells transfected with above plasmid were lysed and the lysates were treated with 45 ug of DNase I before immunoprecipitation with anti-myc antibody. Histone H1 and H2B were detected using specific antibodies (red triangle).

    Techniques Used: Immunoprecipitation, Transfection, Western Blot, Plasmid Preparation

    Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.
    Figure Legend Snippet: Analysis of Fluorescence Resonance Energy Transfer (FRET) between LANA and histones. A. 293T cells were stably transduced with CFP-fused H1myc or CFP-fused H2Bmyc. YFP-LANA-Flag was transfected alone in H1-CFP or H2B-CFP expressing cells. To capture the control images for FRET analysis, H1-CFP and H2B-CFP cells were excited with 405 nm laser to detect any bleed through in YFP emission and also signals in FRET channel. Similarly, YFP-LANA was excited with 515 mm to detect the fluorescence in YFP channel as we all FRET Channel. These individually expressing proteins did not show any signals in FRET channel. B. H1-CFP+ LANA-YFP and H2B-CFP+LANA-YFP expressing cells were excited with 405 nm laser to excite CFP proteins, which emits at 477 nm (cyan). Emission spectra of CFP fall in the excitation range of YFP (514 nm). Therefore, based on the proximity of the proteins to transfer energy from the donor to acceptor, emissions from donor can excite the acceptor and thus there is FRET. Emission from CFP fused with H1 and H2B were able to excite YFP-fused with LANA to emit yellow (535 nm) signals. FRET Channel detected almost similar levels of signals in both H1 and H2B transfected with LANA. Co-localized FRET index or FRET efficiency were calculated ImageJ software, which showed comparable localization of H1 and H2B with LANA. C. Proposed model of LANA’s interaction with histone H1 and H2B fused with CFP. For an efficient FRET the intermolecular distance is deciding factor and the molecules separated by less than 10 nm can transfer energy to yield FRET signals. D. Western blot with anti-myc to show the expressions of CFP-fused H1 and H2B and anti-Flag to show comparable expression of YFP-LANA-Flag in those cells. E: Cells used in FRET assay show binding of LANA with histone H1 and H2B after treatment with DNase I as well as MNase. CFP-H1-myc, and CFP-H2B-myc stably expressing in 293T cells were transfected with Flag vector or YFP-LANA-Flag. Lysates from these cells were divided into three parts, i) untreated, ii) MNase treated, iii) DNase I treated followed by immunoprecipitation of LANA with anti-Flag antibody. Immunoprecipitated LANA in all the set was detected with anti-Flag blot (IB:Flag). Co-precipitating H1 and H2B fused to CFP were detected with anti-myc blot (IB:myc). Detection of H1 and H2B in all three conditions (untreated, MNase and DNase I treated) in LANA expressing cells, but not in vector transfected cells, confirmed specific association of both histones with LANA.

    Techniques Used: Fluorescence, Förster Resonance Energy Transfer, Stable Transfection, Transduction, Transfection, Expressing, Software, Western Blot, Binding Assay, Plasmid Preparation, Immunoprecipitation

    Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).
    Figure Legend Snippet: Localization of GFP-fused histone H1 and H2B with LANA in PEL cells. KSHV uninfected (BJAB) and infected (BCBL1 and JSC1) cells were transduced with lentivirus containing GFP-H1myc and GFP-H2Bmyc (schematic shown above panel A). Transduced cells were selected to obtain pure population of cells expressing GFP fused proteins. GFP-NLSmyc was used as control in this assay. A. Chromosome spreads of BCBL1 with GFP-H1 and GFP-H2B shows staining of entire chromosome with green but not in case of control GFP-NLSmyc. B. Chromosome spreads prepared from JSC1 cells stably expressing GFP-H1 and GFP-H2B. C. Chromosome spreads of BJAB cells expressing GFP-H1 and GFP-H2B. LANA were detected in above chromosome spreads using anti-LANA specific antibody. BCBL1 and JSC1 showed punctate dots throughout the chromosome, as expected. Lack of LANA signals in BJAB (KSHV negative cells) confirmed specificity of LANA detection. GFP-H1+LANA and GFP-H2B+LANA panels show yellow spots on the magnified view of the images. D. BCBL1 cells expressing GFP-NLSmyc, GFP-H1myc and GFP-H2Bmyc were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-Myc antibody. Co-precipitating endogenous LANA was detected using anti-LANA antibody (IB:LANA). GFP and myc fused proteins were detected in anti-myc western blot (IB:myc). E. BJAB and BCBL1 cells stably expressing GFP-H1 or GFP-H2B were lysed and the lysates were treated with DNase I before immunoprecipitation with anti-LANA antibody (LANA-IP panels). Co-precipitating GFP-H1 and GFP-H2B were detected with anti-myc antibody (IB:myc).

    Techniques Used: Infection, Transduction, Expressing, Staining, Stable Transfection, Immunoprecipitation, Western Blot

    33) Product Images from "Protein flexibility is key to cisplatin crosslinking in calmodulin"

    Article Title: Protein flexibility is key to cisplatin crosslinking in calmodulin

    Journal: Protein Science : A Publication of the Protein Society

    doi: 10.1002/pro.2111

    Native ESI spectra of protein complexes. (A) Ca-CaM:melittin = 1:1; (B) (CaM:cisplatin = 1:2)–Ca:melittin = 1:1; (C) (Ca–CaM:cisplatin = 1:2):melittin = 1:1.
    Figure Legend Snippet: Native ESI spectra of protein complexes. (A) Ca-CaM:melittin = 1:1; (B) (CaM:cisplatin = 1:2)–Ca:melittin = 1:1; (C) (Ca–CaM:cisplatin = 1:2):melittin = 1:1.

    Techniques Used: Chick Chorioallantoic Membrane Assay

    34) Product Images from "Genomic Imprinting Controls Matrix Attachment Regions in the Igf2 Gene"

    Article Title: Genomic Imprinting Controls Matrix Attachment Regions in the Igf2 Gene

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.24.8953-8959.2003

    MAR assays. We extracted 10 5 nuclei from a tissue sample with 2 M NaCl. The resulting nuclear halos were digested with restriction enzymes (A). Matrix-bound DNA (pellet, P) was isolated from loop DNA (supernatant, S) by ultrafiltration (see Materials and Methods). The relative enrichment of target sequences in the pellet fraction was determined by real-time quantitative PCR. For allele-specific MAR assays, primers were designed on either side of polymorphic restriction sites and quantifications were performed on DNA from the pellet fraction undigested or digested with the polymorphic enzyme (see Materials and Methods). Alternatively, for precise mapping of nuclear matrix attachments, nuclear halos were digested with DNase I before performing real-time PCR quantifications on the pellet fraction (B). Pictures show nuclei, nuclear halos, and restriction enzyme-treated halos stained with Hoechst. Scale bars, 10 μm.
    Figure Legend Snippet: MAR assays. We extracted 10 5 nuclei from a tissue sample with 2 M NaCl. The resulting nuclear halos were digested with restriction enzymes (A). Matrix-bound DNA (pellet, P) was isolated from loop DNA (supernatant, S) by ultrafiltration (see Materials and Methods). The relative enrichment of target sequences in the pellet fraction was determined by real-time quantitative PCR. For allele-specific MAR assays, primers were designed on either side of polymorphic restriction sites and quantifications were performed on DNA from the pellet fraction undigested or digested with the polymorphic enzyme (see Materials and Methods). Alternatively, for precise mapping of nuclear matrix attachments, nuclear halos were digested with DNase I before performing real-time PCR quantifications on the pellet fraction (B). Pictures show nuclei, nuclear halos, and restriction enzyme-treated halos stained with Hoechst. Scale bars, 10 μm.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Staining

    Matrix association patterns of  Igf2  MARs during liver development. Below the bar graphs, the positions of restriction sites (vertical bars), real-time PCR-amplified sequences (small horizontal bars), potential MARs, and exons (solid boxes) are indicated. (A) Nuclear halos prepared from liver nuclei at the indicated developmental stages were digested with  Xba I,  Hin dIII, and  Bam HI. The graph shows the relative matrix attachment levels, expressed as the ratio between the amount of target sequence in the loop (S) and MAR fractions (P) after normalization to a negative control (NC), which was given the value of 1 (see Materials and Methods). In our assays, highly expressed genes such as  gapdh  and  H19  (not shown) are weakly retained in the matrix fraction relative to the negative control. Error bars show the standard deviation for four (embryonic days 15.5 and 7.5) and two (embryonic day 30) independent experiments. In this experiment, MAR2 enrichment was investigated with the DMR2 primers located in the same restriction fragment. (B) Nuclear halos prepared from 7.5-day-old mouse liver were treated with DNase I. The left panel shows PCR quantifications on matrix-associated DNA, normalized to the  Igκ  MAR. A control performed on total genomic DNA is also shown (right panel). Error bars show the standard deviation for two independent experiments.
    Figure Legend Snippet: Matrix association patterns of Igf2 MARs during liver development. Below the bar graphs, the positions of restriction sites (vertical bars), real-time PCR-amplified sequences (small horizontal bars), potential MARs, and exons (solid boxes) are indicated. (A) Nuclear halos prepared from liver nuclei at the indicated developmental stages were digested with Xba I, Hin dIII, and Bam HI. The graph shows the relative matrix attachment levels, expressed as the ratio between the amount of target sequence in the loop (S) and MAR fractions (P) after normalization to a negative control (NC), which was given the value of 1 (see Materials and Methods). In our assays, highly expressed genes such as gapdh and H19 (not shown) are weakly retained in the matrix fraction relative to the negative control. Error bars show the standard deviation for four (embryonic days 15.5 and 7.5) and two (embryonic day 30) independent experiments. In this experiment, MAR2 enrichment was investigated with the DMR2 primers located in the same restriction fragment. (B) Nuclear halos prepared from 7.5-day-old mouse liver were treated with DNase I. The left panel shows PCR quantifications on matrix-associated DNA, normalized to the Igκ MAR. A control performed on total genomic DNA is also shown (right panel). Error bars show the standard deviation for two independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Sequencing, Negative Control, Standard Deviation, Polymerase Chain Reaction

    35) Product Images from "Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16"

    Article Title: Expression, purification and immunological characterization of the transforming protein E7, from cervical cancer-associated human papillomavirus type 16

    Journal: Clinical and Experimental Immunology

    doi: 10.1046/j.1365-2249.1999.00813.x

    Western blot analysis. Baculovirus-derived E7 protein was subjected to 13.5% SDS–PAGE, and proteins were transferred to nitrocellulose membranes. The membranes were probed with pooled sera from mice immunized with either E7GST/Quil-A (lane 2), ovalbumin/Quil-A (lane 1), E7-specific MoAb (lane 3), or irrelevant MoAb (lane 4). The arrow indicates the position of E7 protein. MW, Molecular weight in kD.
    Figure Legend Snippet: Western blot analysis. Baculovirus-derived E7 protein was subjected to 13.5% SDS–PAGE, and proteins were transferred to nitrocellulose membranes. The membranes were probed with pooled sera from mice immunized with either E7GST/Quil-A (lane 2), ovalbumin/Quil-A (lane 1), E7-specific MoAb (lane 3), or irrelevant MoAb (lane 4). The arrow indicates the position of E7 protein. MW, Molecular weight in kD.

    Techniques Used: Western Blot, Derivative Assay, SDS Page, Mouse Assay, Molecular Weight

    Groups of 10 C57Bl/6J mice were immunized twice at 21-day intervals with either 50 μg E7GST protein and Quil-A (adjuvant) or Quil-A only. Mice were challenged 14 days after the last immunization with 2 × 10 6 cells of a tumour cell line transfected with the E7 protein (C3). Mice were killed 14 days after the tumour challenge and tumour weights were recorded. The scattergram of the data as well as box and whisker plot of the data are shown.
    Figure Legend Snippet: Groups of 10 C57Bl/6J mice were immunized twice at 21-day intervals with either 50 μg E7GST protein and Quil-A (adjuvant) or Quil-A only. Mice were challenged 14 days after the last immunization with 2 × 10 6 cells of a tumour cell line transfected with the E7 protein (C3). Mice were killed 14 days after the tumour challenge and tumour weights were recorded. The scattergram of the data as well as box and whisker plot of the data are shown.

    Techniques Used: Mouse Assay, Transfection, Whisker Assay

    Immunoprecipitation analysis. Baculovirus-derived E7 protein insect cell lysate was treated with pooled sera from mice immunized with either E7GST/Quil-A (lane 2), or ovalbumin/Quil-A (lane 1). The immune complexes were isolated using protein-A Sepharose beads and subjected to SDS–PAGE, along with a direct baculovirus E7 sample (lane 3). The E7 was detected using a mouse MoAb and anti-mouse immunoglobulin horseradish peroxidase. The arrow indicates the position of E7 protein. MW, Molecular weight in kD.
    Figure Legend Snippet: Immunoprecipitation analysis. Baculovirus-derived E7 protein insect cell lysate was treated with pooled sera from mice immunized with either E7GST/Quil-A (lane 2), or ovalbumin/Quil-A (lane 1). The immune complexes were isolated using protein-A Sepharose beads and subjected to SDS–PAGE, along with a direct baculovirus E7 sample (lane 3). The E7 was detected using a mouse MoAb and anti-mouse immunoglobulin horseradish peroxidase. The arrow indicates the position of E7 protein. MW, Molecular weight in kD.

    Techniques Used: Immunoprecipitation, Derivative Assay, Mouse Assay, Isolation, SDS Page, Molecular Weight

    Reactivity of antisera from E7GST/Quil-A-immunized mice (•) as measured by ELISA using plates coated with GF105 synthetic E7 peptide. Quil-A adjuvant only immunized mice (□) were used as control. The sera from each group were pooled and analysed in triplicate at doubling dilutions starting from 1:100. Data shown as mean ± s.e.m. of the optical density (OD) reading at 415 nm for samples in triplicate.
    Figure Legend Snippet: Reactivity of antisera from E7GST/Quil-A-immunized mice (•) as measured by ELISA using plates coated with GF105 synthetic E7 peptide. Quil-A adjuvant only immunized mice (□) were used as control. The sera from each group were pooled and analysed in triplicate at doubling dilutions starting from 1:100. Data shown as mean ± s.e.m. of the optical density (OD) reading at 415 nm for samples in triplicate.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    Reactivity of antisera from E7GST/Quil-A-immunized mice (•) as measured by ELISA using plates coated with E7MS2 protein. Quil-A adjuvant only immunized mice (□) were used as control. The sera from each group were pooled and analysed in triplicate at doubling dilutions starting from 1:100. Data shown as mean ± s.e.m. of the optical density (OD) reading at 415 nm for samples in triplicate.
    Figure Legend Snippet: Reactivity of antisera from E7GST/Quil-A-immunized mice (•) as measured by ELISA using plates coated with E7MS2 protein. Quil-A adjuvant only immunized mice (□) were used as control. The sera from each group were pooled and analysed in triplicate at doubling dilutions starting from 1:100. Data shown as mean ± s.e.m. of the optical density (OD) reading at 415 nm for samples in triplicate.

    Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

    36) Product Images from "Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair"

    Article Title: Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2005595

    Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR locus. Bisulphite-converted gDNA was subjected to Illumina amplicon sequencing across a region spanning 13 CpG dinucleotides ( S1A Fig ), and reads were classified according to the proportion of nonconverted (methylated) CpGs. The black dashed line indicates the expected level of methylation across all alleles when imprinting is completely maintained (50%). In subsequent editing experiments, the percentage of hypermethylated ( > 80%) strands is reported together with histograms showing allele-specific mutation frequency. Quantitative data underlying panel D are provided in S1 Data , and details of MiSeq libraries including SRA accessions are provided in S2 Data . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.
    Figure Legend Snippet: Imprinted chromatin as a model system to quantify epigenetic influences on genome editing. (A) Schematic outlining the experimental workflow. Throughout the text, F1 hybrid cell lines are depicted with the maternal strain denoted before the paternal strain (i.e., In B×J: B is maternal and J paternal). sgRNAs are designed to cleave approximately 40–100 bp from a heterozygous SNP within imprinted chromatin (open and closed circles). MiSeq amplicons span both the SNP and site of mutation, which allows simultaneous assessment of genome editing outcome and parental allele at high-throughput. (B) Top: schematic showing the imprinted mouse Kcnq1 gene including H3K9me3 ChIP and DNase-I–seq data from mESCs available through EncODE (ENCSR000CFZ, GSM1014187) (bottom). Higher-resolution view of the KvDMR imprinted CpG island within Kcnq1 , showing the position of three sgRNAs used in panel E. (C) Allele-specific enrichment of H3K9me3 and H4K20me3. PCR fragments spanning the target sites of sgKvDMR#2 and #3 were amplified from input, or ChIP DNA prior to Sanger sequencing across an allelic SNP. gDNA = genomic DNA from purebred mice. (D) Example of CpG methylation data from the KvDMR locus. Bisulphite-converted gDNA was subjected to Illumina amplicon sequencing across a region spanning 13 CpG dinucleotides ( S1A Fig ), and reads were classified according to the proportion of nonconverted (methylated) CpGs. The black dashed line indicates the expected level of methylation across all alleles when imprinting is completely maintained (50%). In subsequent editing experiments, the percentage of hypermethylated ( > 80%) strands is reported together with histograms showing allele-specific mutation frequency. Quantitative data underlying panel D are provided in S1 Data , and details of MiSeq libraries including SRA accessions are provided in S2 Data . ChIP, chromatin immunoprecipitation; gDNA, genomic DNA; HDR, homology-directed repair; mESC, mouse embryonic stem cell, NHEJ, nonhomologous end joining; sgRNA, single guide RNA; SNP, single nucleotide polymorphism; SRA, Sequence Read Archive; ssODN, single-stranded oligodeoxynucleotide.

    Techniques Used: Mutagenesis, High Throughput Screening Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Sequencing, Mouse Assay, CpG Methylation Assay, Methylation, Non-Homologous End Joining

    37) Product Images from "Detecting and minimizing zinc contamination in physiological solutions"

    Article Title: Detecting and minimizing zinc contamination in physiological solutions

    Journal: BMC Physiology

    doi: 10.1186/1472-6793-4-4

    Detecting elevations as low as 100 pM Zn with FluoZin-3 . The solution contained 500 nM FluoZin-3 and 10 μM Ca-EDTA in Hepes buffered saline. 'H 2 O' indicates the addition of 2 μl water. The trace was subjected to a 7 point Savitzky-Golay smoothing procedure. The solid line is the mean of the data acquired between 0 and 2 min, and the dotted lines the SDs.
    Figure Legend Snippet: Detecting elevations as low as 100 pM Zn with FluoZin-3 . The solution contained 500 nM FluoZin-3 and 10 μM Ca-EDTA in Hepes buffered saline. 'H 2 O' indicates the addition of 2 μl water. The trace was subjected to a 7 point Savitzky-Golay smoothing procedure. The solid line is the mean of the data acquired between 0 and 2 min, and the dotted lines the SDs.

    Techniques Used:

    Zinc release from a bipolar stainless steel electrode . '+' indicates the beginning of current passage with the bottom electrode as the anode, while '-' denotes that the polarity was reversed. A 100 Hz square wave with pulse duration of 30 μs was applied for 2.5 s at a current amplitude of 500 μA. The experiment was performed in Hepes saline with 2μM NPG and 50 μM Ca-EDTA. Pseudocolor images are the differences between the fluorescence at the peak of the response and that immediately prior to delivering the stimulus; Left '+' stimulus, right '-' stimulus. Color bar 0–50. Scale bar 100 μm.
    Figure Legend Snippet: Zinc release from a bipolar stainless steel electrode . '+' indicates the beginning of current passage with the bottom electrode as the anode, while '-' denotes that the polarity was reversed. A 100 Hz square wave with pulse duration of 30 μs was applied for 2.5 s at a current amplitude of 500 μA. The experiment was performed in Hepes saline with 2μM NPG and 50 μM Ca-EDTA. Pseudocolor images are the differences between the fluorescence at the peak of the response and that immediately prior to delivering the stimulus; Left '+' stimulus, right '-' stimulus. Color bar 0–50. Scale bar 100 μm.

    Techniques Used: Fluorescence

    Titration of FluoZin-3 with Zn in the presence and absence of Ca-EDTA . The ZnSO 4 additions beginning at 2 minutes and at 2 minute intervals were 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1, 3.2 and 10 μM. 500 nM FluoZin-3 in Hepes saline at 26°C.
    Figure Legend Snippet: Titration of FluoZin-3 with Zn in the presence and absence of Ca-EDTA . The ZnSO 4 additions beginning at 2 minutes and at 2 minute intervals were 0.001, 0.0032, 0.01, 0.032, 0.1, 0.32, 1, 3.2 and 10 μM. 500 nM FluoZin-3 in Hepes saline at 26°C.

    Techniques Used: Titration

    Stirring induces the slow release of zinc from methacrylate cuvettes . The Hepes saline solution contained 500 nM FluoZin-3 in Hepes saline.
    Figure Legend Snippet: Stirring induces the slow release of zinc from methacrylate cuvettes . The Hepes saline solution contained 500 nM FluoZin-3 in Hepes saline.

    Techniques Used:

    38) Product Images from "The MogR Transcriptional Repressor Regulates Nonhierarchal Expression of Flagellar Motility Genes and Virulence in Listeria monocytogenes"

    Article Title: The MogR Transcriptional Repressor Regulates Nonhierarchal Expression of Flagellar Motility Genes and Virulence in Listeria monocytogenes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0020030

    Purified MogR Is Sufficient to Bind the Promoter Regions of Multiple Flagellar Motility Genes (A) Gel shift analysis of MogR binding to the flaA promoter region. Radiolabeled flaA promoter DNA spanning region −162 to +8 relative to the transcription start site was incubated with increasing concentrations of purified His 6 -tagged MogR (lanes 1 to 8) and in the presence of unlabeled competitor DNA (lanes 7 and 8). The binding reactions were analyzed by nondenaturing PAGE. The identity of unlabeled competitor DNA added in 50-fold excess to binding reactions is indicated. Shifted (S), supershifted (SS), and super-supershifted (SSS) DNA complexes are indicated. (B) Gel shift analysis of MogR binding to flagellar motility gene promoter regions. Increasing amounts of purified His 6 -tagged MogR were incubated with various radiolabeled DNA probes (lanes 1 to 4) and in the presence of unlabeled competitor probe DNA of the same identity (lane 4). The identity of each probe is indicated. The probe for cheY spans the region from −108 to +74 relative to the transcription start site. The probe for lmo0703 spans the region from −164 to +21 relative to the translation start site. The probes for lmo0723 and lmo1699 span the regions from −123 to +18 relative to the translation start site. (C) DNase I footprinting analysis of MogR binding to the flaA promoter region. The noncoding strand probe spans the region from −162 to +8 relative to the transcription start site. Negative numbers indicate the approximate distance from the transcriptional start site. The high affinity MogR binding site is indicated by a solid bracket and is denoted as region I. The lower affinity binding sites are marked with dashed brackets and identified as regions II and III. (D) DNA sequence of the flaA promoter region. The bottom strand is the coding strand and reads 5′ to 3′. The −35 and −10 elements and transcription start site (+1) are indicated in bold and by a solid line above the sequence. Solid bracket indicates the region protected at low MogR concentrations (region I), while the dashed brackets mark the regions protected with higher concentrations of MogR (regions II and III).
    Figure Legend Snippet: Purified MogR Is Sufficient to Bind the Promoter Regions of Multiple Flagellar Motility Genes (A) Gel shift analysis of MogR binding to the flaA promoter region. Radiolabeled flaA promoter DNA spanning region −162 to +8 relative to the transcription start site was incubated with increasing concentrations of purified His 6 -tagged MogR (lanes 1 to 8) and in the presence of unlabeled competitor DNA (lanes 7 and 8). The binding reactions were analyzed by nondenaturing PAGE. The identity of unlabeled competitor DNA added in 50-fold excess to binding reactions is indicated. Shifted (S), supershifted (SS), and super-supershifted (SSS) DNA complexes are indicated. (B) Gel shift analysis of MogR binding to flagellar motility gene promoter regions. Increasing amounts of purified His 6 -tagged MogR were incubated with various radiolabeled DNA probes (lanes 1 to 4) and in the presence of unlabeled competitor probe DNA of the same identity (lane 4). The identity of each probe is indicated. The probe for cheY spans the region from −108 to +74 relative to the transcription start site. The probe for lmo0703 spans the region from −164 to +21 relative to the translation start site. The probes for lmo0723 and lmo1699 span the regions from −123 to +18 relative to the translation start site. (C) DNase I footprinting analysis of MogR binding to the flaA promoter region. The noncoding strand probe spans the region from −162 to +8 relative to the transcription start site. Negative numbers indicate the approximate distance from the transcriptional start site. The high affinity MogR binding site is indicated by a solid bracket and is denoted as region I. The lower affinity binding sites are marked with dashed brackets and identified as regions II and III. (D) DNA sequence of the flaA promoter region. The bottom strand is the coding strand and reads 5′ to 3′. The −35 and −10 elements and transcription start site (+1) are indicated in bold and by a solid line above the sequence. Solid bracket indicates the region protected at low MogR concentrations (region I), while the dashed brackets mark the regions protected with higher concentrations of MogR (regions II and III).

    Techniques Used: Purification, Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Footprinting, Sequencing

    MogR Recognizes a Minimum of Two TTTT-N 5 -AAAA Sites (A) Gel shift analysis of MogR binding to the −35 region of the flaA promoter. Radiolabeled flaA probe DNA spanning region −58 to −13 relative to the transcription start site was incubated with increasing concentrations of purified His 6 -tagged MogR (lanes 2 to 4, 6 to 8, and 10 to 12) and in the presence of unlabeled competitor DNA (lanes 4, 8, and 12). Radiolabeled wild-type (wt) probe was used in lanes 1 to 4; radiolabeled probe harboring mutations in four residues of one binding site (TA) was used in lanes 5 to 8; and a radiolabeled probe harboring mutations in six residues interspersed between the predicted MogR contact sites (N-mut) was used in lanes 9 to 12. Sequences of the DNA probes are given below the panel. Mutations introduced are underlined and in bold. Gray boxes denote putative MogR binding sites. The binding reactions were analyzed by nondenaturing PAGE. The identity of unlabeled competitor DNA added in 50-fold excess to binding reactions is indicated. (B) Sequences of predicted MogR binding sites within the promoter regions of genes directly regulated by MogR. Gray boxes around text in bold indicates predicted MogR binding sites with italicized text indicating deviations from the consensus TTTT-N 5 -AAAA binding site. The transcriptional start sites of flaA, cheY, and lmo0675 have been precisely mapped by primer extension and are designated by +1. The approximate transcriptional start regions for lmo0723, lmo0703, and lmo1699 have been identified by primer extension. The predicted −35 and −10 elements are underlined . The distance to the coding region is indicated, and the translation initiation codon is boxed and in bold. Region I, and regions II and III correspond to the high and low affinity binding sites, respectively, mapped by DNase I footprinting. Region I is denoted by a solid bracket. Regions II and III are marked by a dashed bracket.
    Figure Legend Snippet: MogR Recognizes a Minimum of Two TTTT-N 5 -AAAA Sites (A) Gel shift analysis of MogR binding to the −35 region of the flaA promoter. Radiolabeled flaA probe DNA spanning region −58 to −13 relative to the transcription start site was incubated with increasing concentrations of purified His 6 -tagged MogR (lanes 2 to 4, 6 to 8, and 10 to 12) and in the presence of unlabeled competitor DNA (lanes 4, 8, and 12). Radiolabeled wild-type (wt) probe was used in lanes 1 to 4; radiolabeled probe harboring mutations in four residues of one binding site (TA) was used in lanes 5 to 8; and a radiolabeled probe harboring mutations in six residues interspersed between the predicted MogR contact sites (N-mut) was used in lanes 9 to 12. Sequences of the DNA probes are given below the panel. Mutations introduced are underlined and in bold. Gray boxes denote putative MogR binding sites. The binding reactions were analyzed by nondenaturing PAGE. The identity of unlabeled competitor DNA added in 50-fold excess to binding reactions is indicated. (B) Sequences of predicted MogR binding sites within the promoter regions of genes directly regulated by MogR. Gray boxes around text in bold indicates predicted MogR binding sites with italicized text indicating deviations from the consensus TTTT-N 5 -AAAA binding site. The transcriptional start sites of flaA, cheY, and lmo0675 have been precisely mapped by primer extension and are designated by +1. The approximate transcriptional start regions for lmo0723, lmo0703, and lmo1699 have been identified by primer extension. The predicted −35 and −10 elements are underlined . The distance to the coding region is indicated, and the translation initiation codon is boxed and in bold. Region I, and regions II and III correspond to the high and low affinity binding sites, respectively, mapped by DNase I footprinting. Region I is denoted by a solid bracket. Regions II and III are marked by a dashed bracket.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Incubation, Purification, Polyacrylamide Gel Electrophoresis, Footprinting

    39) Product Images from "SOS Regulation of the Type III Secretion System of Enteropathogenic Escherichia coli ▿"

    Article Title: SOS Regulation of the Type III Secretion System of Enteropathogenic Escherichia coli ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01859-06

    DNase I protection assays to establish purified LexA protein binding in LEE and recA regulatory DNA fragments in vitro. (A) The region of protection from DNase I cleavage, demonstrating LexA binding at the LEE2 / LEE3 promoters, is indicated by a vertical
    Figure Legend Snippet: DNase I protection assays to establish purified LexA protein binding in LEE and recA regulatory DNA fragments in vitro. (A) The region of protection from DNase I cleavage, demonstrating LexA binding at the LEE2 / LEE3 promoters, is indicated by a vertical

    Techniques Used: Purification, Protein Binding, In Vitro, Binding Assay

    40) Product Images from "An epiblast stem cell-derived multipotent progenitor population for axial extension"

    Article Title: An epiblast stem cell-derived multipotent progenitor population for axial extension

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.168187

    Comparison between in vitro protocols to produce NMP-like cells. ) and did not split/passage the cells, which were grown for 5†days in the same flask in the neural or mesodermal conditions. We named the resulting populations ES-neuro/ES-neuroF and ES-meso/ES-mesoF for those with an ES-NMP/ES-NMPF origin, and Epi-neuro and Epi-meso for those with an Epi-NMP origin. (C) Confocal immunofluorescent images of EpiSCs, and ES-NMP, ES-NMPF and Epi-NMP cultures on their 3rd day, and an Epi-meso culture on its 2nd day. Hoechst (nuclei) is in grey, Oct4 in red, Sox2 in green and T in magenta. The composite image of Sox2 (green) and T (magenta) is presented on the right-hand side. (D) Quantification plots of the fluorescence intensity in arbitrary units (a.u.) representing the protein levels in a cell. Each point represents a cell. The x -axis and y -axis represent the fluorescence intensity of either Sox2, T and Oct4. The numbers next to the T versus Sox2 plot indicate the percentage of cells that co-express Sox2 and T under the different conditions. The mean and the coefficient of variation (CV) of the distribution of Sox2, T and Oct4 across the different conditions are presented on the right-hand side: EpiSCs in orange, ES-NMP in yellow, ES-NMPF in turquoise, Epi-NMP in purple and Epi-meso in green.
    Figure Legend Snippet: Comparison between in vitro protocols to produce NMP-like cells. ) and did not split/passage the cells, which were grown for 5†days in the same flask in the neural or mesodermal conditions. We named the resulting populations ES-neuro/ES-neuroF and ES-meso/ES-mesoF for those with an ES-NMP/ES-NMPF origin, and Epi-neuro and Epi-meso for those with an Epi-NMP origin. (C) Confocal immunofluorescent images of EpiSCs, and ES-NMP, ES-NMPF and Epi-NMP cultures on their 3rd day, and an Epi-meso culture on its 2nd day. Hoechst (nuclei) is in grey, Oct4 in red, Sox2 in green and T in magenta. The composite image of Sox2 (green) and T (magenta) is presented on the right-hand side. (D) Quantification plots of the fluorescence intensity in arbitrary units (a.u.) representing the protein levels in a cell. Each point represents a cell. The x -axis and y -axis represent the fluorescence intensity of either Sox2, T and Oct4. The numbers next to the T versus Sox2 plot indicate the percentage of cells that co-express Sox2 and T under the different conditions. The mean and the coefficient of variation (CV) of the distribution of Sox2, T and Oct4 across the different conditions are presented on the right-hand side: EpiSCs in orange, ES-NMP in yellow, ES-NMPF in turquoise, Epi-NMP in purple and Epi-meso in green.

    Techniques Used: In Vitro, Fluorescence

    Related Articles

    Irradiation:

    Article Title: Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides
    Article Snippet: .. All samples were irradiated at 365 nm for 30 min and reaction products were then treated in one of the three ways. (i) Direct analysis: samples were precipitated in ethanol. (ii) Piperidine cleavage reactions: piperidine (100 µl of a 1 M solution) was added to the reaction products and incubated at 90°C for 30 min. Piperidine was removed by lyophilization. (iii) DNase I footprinting: samples were digested with 1 µl of DNase I (final concentration 0.03 mg/ml, Sigma) diluted in 1 mM MgCl2 , 1 mM MnCl2 and 20 mM NaCl (pH 7.3). ..

    Amplification:

    Article Title: A Regulatory Network Controls cabABC Expression Leading to Biofilm and Rugose Colony Development in Vibrio vulnificus
    Article Snippet: .. Protein Purification, EMSA, and DNase I Protection Assay The ORFs of brpT and brpS were amplified by PCR using pairs of primers BRPT04-F and -R or BRPS04-F and -R ( ) and subcloned into pET-28a(+) (Novagen, Madison, WI, United States), resulting in pSH1819 and pSH1823 , respectively. .. The His6 -tagged BrpT and BrpS were expressed in E. coli BL21 (DE3) and purified by affinity chromatography using Ni-NTA agarose (Qiagen).

    In Vitro:

    Article Title: A Regulatory Potential of the Xist Gene Promoter in Vole M. rossiaemeridionalis
    Article Snippet: .. Preparation of Nuclear Extracts and DNase I in vitro Footprinting Nuclear extracts from liver cells and fibroblasts (line sd10) of M. rossiaemeridionalis were isolated as described , and using a CelLytic™ NuCLEAR™ Extraction kit (Sigma). ..

    Chromatography:

    Article Title: Human lysosomal DNase II? contains two requisite PLD-signature (HxK) motifs: Evidence for a pseudodimeric structure of the active enzyme species
    Article Snippet: .. From the lysate supernatant, the protein of interest was purified according to the affinity matrix used: DNase IIα with a Flag-His6 tandem tag was purified by either Flag-affinity chromatography using EZview Red ANTI-FLAG M2 Affinity Gel (Sigma) or IMAC using magnetic Ni2+ -NTA particles (see below); whereas for DNase IIα fused to the hemagglutinin-epitope tag, HA-affinity chromatography using EZview Red ANTI-HA Affinity Gel (Sigma) was performed. ..

    Isolation:

    Article Title: A Regulatory Potential of the Xist Gene Promoter in Vole M. rossiaemeridionalis
    Article Snippet: .. Preparation of Nuclear Extracts and DNase I in vitro Footprinting Nuclear extracts from liver cells and fibroblasts (line sd10) of M. rossiaemeridionalis were isolated as described , and using a CelLytic™ NuCLEAR™ Extraction kit (Sigma). ..

    Footprinting:

    Article Title: The GCN4 bZIP can bind to noncognate gene regulatory sequences
    Article Snippet: .. The DNase I footprinting reactions (20 μl total volume) were assembled by adding bZIP protein to a mixture containing TKMC buffer (20 mM Tris, pH 7.5, 4 mM KCl, 2 mM MgCl2 , 1 mM CaCl2 ), 100 μg/ml BSA (non-acetylated, DNase/RNase/protease-free, Sigma), 5 mM DTT, 1 μg/ml poly(d I –d C ) (Sigma), and 5% glycerol. ..

    Article Title: Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides
    Article Snippet: .. All samples were irradiated at 365 nm for 30 min and reaction products were then treated in one of the three ways. (i) Direct analysis: samples were precipitated in ethanol. (ii) Piperidine cleavage reactions: piperidine (100 µl of a 1 M solution) was added to the reaction products and incubated at 90°C for 30 min. Piperidine was removed by lyophilization. (iii) DNase I footprinting: samples were digested with 1 µl of DNase I (final concentration 0.03 mg/ml, Sigma) diluted in 1 mM MgCl2 , 1 mM MnCl2 and 20 mM NaCl (pH 7.3). ..

    Article Title: A Regulatory Potential of the Xist Gene Promoter in Vole M. rossiaemeridionalis
    Article Snippet: .. Preparation of Nuclear Extracts and DNase I in vitro Footprinting Nuclear extracts from liver cells and fibroblasts (line sd10) of M. rossiaemeridionalis were isolated as described , and using a CelLytic™ NuCLEAR™ Extraction kit (Sigma). ..

    Mouse Assay:

    Article Title: A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse ?-globin locus
    Article Snippet: .. In the case of DNase I hypersensitivity mapping of mouse spleen and liver, mice were treated with N- acetylphenylhydrazine (4 mg/ml in water; Sigma A4626) to induce erythropoiesis of the spleen; 0.04 mg/g body weight was injected intraperitoneally on days 1, 2, and 3, followed by sacrifice on day 5. .. In the case of mutant mice that retained the marker gene, spleen weight increased to 525 ± 25 mg.

    Protein Purification:

    Article Title: A Regulatory Network Controls cabABC Expression Leading to Biofilm and Rugose Colony Development in Vibrio vulnificus
    Article Snippet: .. Protein Purification, EMSA, and DNase I Protection Assay The ORFs of brpT and brpS were amplified by PCR using pairs of primers BRPT04-F and -R or BRPS04-F and -R ( ) and subcloned into pET-28a(+) (Novagen, Madison, WI, United States), resulting in pSH1819 and pSH1823 , respectively. .. The His6 -tagged BrpT and BrpS were expressed in E. coli BL21 (DE3) and purified by affinity chromatography using Ni-NTA agarose (Qiagen).

    Concentration Assay:

    Article Title: Interferon-?-induced chromatin remodeling at the CIITA locus is BRG1 dependent
    Article Snippet: .. DNase I stock (4 mg/ml; Sigma Molecular Biology Grade D5793) was diluted in 1× RSB to a final concentration of 80 ng/ml. ..

    Article Title: Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides
    Article Snippet: .. All samples were irradiated at 365 nm for 30 min and reaction products were then treated in one of the three ways. (i) Direct analysis: samples were precipitated in ethanol. (ii) Piperidine cleavage reactions: piperidine (100 µl of a 1 M solution) was added to the reaction products and incubated at 90°C for 30 min. Piperidine was removed by lyophilization. (iii) DNase I footprinting: samples were digested with 1 µl of DNase I (final concentration 0.03 mg/ml, Sigma) diluted in 1 mM MgCl2 , 1 mM MnCl2 and 20 mM NaCl (pH 7.3). ..

    Incubation:

    Article Title: Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides
    Article Snippet: .. All samples were irradiated at 365 nm for 30 min and reaction products were then treated in one of the three ways. (i) Direct analysis: samples were precipitated in ethanol. (ii) Piperidine cleavage reactions: piperidine (100 µl of a 1 M solution) was added to the reaction products and incubated at 90°C for 30 min. Piperidine was removed by lyophilization. (iii) DNase I footprinting: samples were digested with 1 µl of DNase I (final concentration 0.03 mg/ml, Sigma) diluted in 1 mM MgCl2 , 1 mM MnCl2 and 20 mM NaCl (pH 7.3). ..

    Positron Emission Tomography:

    Article Title: A Regulatory Network Controls cabABC Expression Leading to Biofilm and Rugose Colony Development in Vibrio vulnificus
    Article Snippet: .. Protein Purification, EMSA, and DNase I Protection Assay The ORFs of brpT and brpS were amplified by PCR using pairs of primers BRPT04-F and -R or BRPS04-F and -R ( ) and subcloned into pET-28a(+) (Novagen, Madison, WI, United States), resulting in pSH1819 and pSH1823 , respectively. .. The His6 -tagged BrpT and BrpS were expressed in E. coli BL21 (DE3) and purified by affinity chromatography using Ni-NTA agarose (Qiagen).

    Polymerase Chain Reaction:

    Article Title: A Regulatory Network Controls cabABC Expression Leading to Biofilm and Rugose Colony Development in Vibrio vulnificus
    Article Snippet: .. Protein Purification, EMSA, and DNase I Protection Assay The ORFs of brpT and brpS were amplified by PCR using pairs of primers BRPT04-F and -R or BRPS04-F and -R ( ) and subcloned into pET-28a(+) (Novagen, Madison, WI, United States), resulting in pSH1819 and pSH1823 , respectively. .. The His6 -tagged BrpT and BrpS were expressed in E. coli BL21 (DE3) and purified by affinity chromatography using Ni-NTA agarose (Qiagen).

    Injection:

    Article Title: A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse ?-globin locus
    Article Snippet: .. In the case of DNase I hypersensitivity mapping of mouse spleen and liver, mice were treated with N- acetylphenylhydrazine (4 mg/ml in water; Sigma A4626) to induce erythropoiesis of the spleen; 0.04 mg/g body weight was injected intraperitoneally on days 1, 2, and 3, followed by sacrifice on day 5. .. In the case of mutant mice that retained the marker gene, spleen weight increased to 525 ± 25 mg.

    Purification:

    Article Title: Human lysosomal DNase II? contains two requisite PLD-signature (HxK) motifs: Evidence for a pseudodimeric structure of the active enzyme species
    Article Snippet: .. From the lysate supernatant, the protein of interest was purified according to the affinity matrix used: DNase IIα with a Flag-His6 tandem tag was purified by either Flag-affinity chromatography using EZview Red ANTI-FLAG M2 Affinity Gel (Sigma) or IMAC using magnetic Ni2+ -NTA particles (see below); whereas for DNase IIα fused to the hemagglutinin-epitope tag, HA-affinity chromatography using EZview Red ANTI-HA Affinity Gel (Sigma) was performed. ..

    other:

    Article Title: Fibronectin-binding protein B (FnBPB) from Staphylococcus aureus protects against the antimicrobial activity of histones
    Article Snippet: Protease-free DNase I, BSA (BSA), skim milk, CTH, and human histones H1, H2A, H2B, H3, and H4 were purchased from Sigma.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 88
    Millipore tppe digestion buffer
    <t>TPPE</t> cleaves the reduced pro-BTH6 in the thionin domain. LC-ESI-MS analysis of the digestion of <t>carboxymethylated</t> myc-proBTH6-strep with recombinant TPPE. The acidic domain is cleaved several times with additional sites compared with the oxidized proprotein.
    Tppe Digestion Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tppe digestion buffer/product/Millipore
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tppe digestion buffer - by Bioz Stars, 2020-09
    88/100 stars
      Buy from Supplier

    90
    Millipore cytokine homogenate buffer
    Leptin and <t>cytokine</t> levels (IL-6 and TNF- α ) after 4 and 6 weeks on the diets. Consistent with the weight gain and epididymal fat pad weights, leptin levels were significantly higher after 4 and 6 weeks in the HAGE-HF group compared to the other groups. Also consistent with the liver histology and presence of inflammation after 6 weeks on the diet, IL-6 and TNF- α levels were significantly higher in the HAGE-HF group after 6 weeks on the diet.
    Cytokine Homogenate Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cytokine homogenate buffer/product/Millipore
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cytokine homogenate buffer - by Bioz Stars, 2020-09
    90/100 stars
      Buy from Supplier

    Image Search Results


    TPPE cleaves the reduced pro-BTH6 in the thionin domain. LC-ESI-MS analysis of the digestion of carboxymethylated myc-proBTH6-strep with recombinant TPPE. The acidic domain is cleaved several times with additional sites compared with the oxidized proprotein.

    Journal: The Journal of Biological Chemistry

    Article Title: Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley *

    doi: 10.1074/jbc.M115.647859

    Figure Lengend Snippet: TPPE cleaves the reduced pro-BTH6 in the thionin domain. LC-ESI-MS analysis of the digestion of carboxymethylated myc-proBTH6-strep with recombinant TPPE. The acidic domain is cleaved several times with additional sites compared with the oxidized proprotein.

    Article Snippet: The reduced and cysteine-carboxymethylated protein samples were dialyzed against TPPE digestion buffer (25 m m MES, 100 m m NaCl, 10 m m CaCl2 , pH 6.5) using Amicon Ultra 10K ultrafiltration centrifugal filters (Millipore) according to the recommendations of the manufacturer.

    Techniques: Mass Spectrometry, Recombinant

    Leptin and cytokine levels (IL-6 and TNF- α ) after 4 and 6 weeks on the diets. Consistent with the weight gain and epididymal fat pad weights, leptin levels were significantly higher after 4 and 6 weeks in the HAGE-HF group compared to the other groups. Also consistent with the liver histology and presence of inflammation after 6 weeks on the diet, IL-6 and TNF- α levels were significantly higher in the HAGE-HF group after 6 weeks on the diet.

    Journal: BioMed Research International

    Article Title: Advanced Glycation End Products Induce Obesity and Hepatosteatosis in CD-1 Wild-Type Mice

    doi: 10.1155/2016/7867852

    Figure Lengend Snippet: Leptin and cytokine levels (IL-6 and TNF- α ) after 4 and 6 weeks on the diets. Consistent with the weight gain and epididymal fat pad weights, leptin levels were significantly higher after 4 and 6 weeks in the HAGE-HF group compared to the other groups. Also consistent with the liver histology and presence of inflammation after 6 weeks on the diet, IL-6 and TNF- α levels were significantly higher in the HAGE-HF group after 6 weeks on the diet.

    Article Snippet: The remaining liver was washed with cold HBSS and suspended in cytokine homogenate buffer (150 mM NaCl, 15 mM Tris, 1 mM CaCl2 ·2H2 O, and 1 mM MgCl2 ·6H2 O, adjusted to pH 7.4) plus 100x protease inhibitor cocktail 1 (Calbiochem, La Jolla, California) to a total volume of 10 mL and homogenized on ice using a Polytron® Homogenizer (Kinematica Inc., Bohemia, NY).

    Techniques: