fanci  (Bethyl)

 
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  • 88
    Name:
    Gemin5 Antibody AbVantage Pack
    Description:

    Catalog Number:
    A310-454A
    Price:
    [472.0]
    Applications:
    Western Blot,Immunoprecipitation
    Host:
    Rabbit
    Conjugate:
    Unconjugated
    Size:
    1 kit
    Category:
    Antibody
    Antibody Type:
    Primary antibody
    Isotype:
    IgG
    Reactivity:
    Human
    Buy from Supplier


    Structured Review

    Bethyl fanci
    The UBL5-binding ability of <t>FANCI</t> is required for FA pathway functionality Representative images of HeLa cells transfected with GFP-tagged FANCI WT or ∆UBL5 and treated with 0.2 μM MMC for 12 h. Scale bar, 10 μm. Quantification of data in (A). Results from three independent experiments (mean ± SD) are shown. More than 200 cells were counted for each condition. Quantification of cells containing <t>FANCD2</t> foci. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 transfected with siRNA targeting the 5′-UTR region of FANCI were treated with 0.2 μM MMC for 16 h, fixed and analyzed for FANCD2 foci formation by immunostaining with FANCD2 antibody. Cells containing more than five foci were scored as foci positive. Results from three independent experiments (mean ± SD) are shown. More than 300 cells were counted for each condition. Quantification of chromosomal aberrations. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 transfected with siRNA targeting the 5′-UTR region of FANCI were treated with 120 nM MMC for 24 h, then treated with nocodazole for additional 2 h and collected. Metaphase spreads were prepared and chromosomal aberrations were quantified. Black lines indicate the mean of the data plotted. P -values were calculated using Mann–Whitney U -test ( n = 50). Model of UBL5 function in the FA pathway (see main text for details). Ub, ubiquitin.

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    fanci - by Bioz Stars, 2020-09
    88/100 stars

    Images

    1) Product Images from "Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway"

    Article Title: Ubiquitin-like protein UBL5 promotes the functional integrity of the Fanconi anemia pathway

    Journal: The EMBO Journal

    doi: 10.15252/embj.201490376

    The UBL5-binding ability of FANCI is required for FA pathway functionality Representative images of HeLa cells transfected with GFP-tagged FANCI WT or ∆UBL5 and treated with 0.2 μM MMC for 12 h. Scale bar, 10 μm. Quantification of data in (A). Results from three independent experiments (mean ± SD) are shown. More than 200 cells were counted for each condition. Quantification of cells containing FANCD2 foci. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 transfected with siRNA targeting the 5′-UTR region of FANCI were treated with 0.2 μM MMC for 16 h, fixed and analyzed for FANCD2 foci formation by immunostaining with FANCD2 antibody. Cells containing more than five foci were scored as foci positive. Results from three independent experiments (mean ± SD) are shown. More than 300 cells were counted for each condition. Quantification of chromosomal aberrations. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 transfected with siRNA targeting the 5′-UTR region of FANCI were treated with 120 nM MMC for 24 h, then treated with nocodazole for additional 2 h and collected. Metaphase spreads were prepared and chromosomal aberrations were quantified. Black lines indicate the mean of the data plotted. P -values were calculated using Mann–Whitney U -test ( n = 50). Model of UBL5 function in the FA pathway (see main text for details). Ub, ubiquitin.
    Figure Legend Snippet: The UBL5-binding ability of FANCI is required for FA pathway functionality Representative images of HeLa cells transfected with GFP-tagged FANCI WT or ∆UBL5 and treated with 0.2 μM MMC for 12 h. Scale bar, 10 μm. Quantification of data in (A). Results from three independent experiments (mean ± SD) are shown. More than 200 cells were counted for each condition. Quantification of cells containing FANCD2 foci. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 transfected with siRNA targeting the 5′-UTR region of FANCI were treated with 0.2 μM MMC for 16 h, fixed and analyzed for FANCD2 foci formation by immunostaining with FANCD2 antibody. Cells containing more than five foci were scored as foci positive. Results from three independent experiments (mean ± SD) are shown. More than 300 cells were counted for each condition. Quantification of chromosomal aberrations. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 transfected with siRNA targeting the 5′-UTR region of FANCI were treated with 120 nM MMC for 24 h, then treated with nocodazole for additional 2 h and collected. Metaphase spreads were prepared and chromosomal aberrations were quantified. Black lines indicate the mean of the data plotted. P -values were calculated using Mann–Whitney U -test ( n = 50). Model of UBL5 function in the FA pathway (see main text for details). Ub, ubiquitin.

    Techniques Used: Binding Assay, Transfection, Stable Transfection, Expressing, Immunostaining, MANN-WHITNEY

    FANCI forms UBL5-dependent homomeric complexes HEK293T cells transfected with non-targeting control or FANCD2 siRNA were co-transfected with HA-FANCI and GFP-FANCI constructs as indicated. Whole-cell extracts (WCE) were subjected to GFP immunoprecipitation followed by immunoblotting with the indicated antibodies. Extracts of HEK293T cells co-transfected with indicated combinations of HA-FANCI and GFP-FANCI constructs were subjected to GFP immunoprecipitation followed by immunoblotting with HA and GFP antibodies. U2OS cells were transfected with non-targeting control (CTRL) or UBL5 siRNA, subsequently transfected with a construct encoding DmrB-GFP-FANCI and then treated with 100 nM AP20187 (B/B homodimerizer) or left untreated. Cell extracts were analyzed by immunoblotting with the indicated antibodies.
    Figure Legend Snippet: FANCI forms UBL5-dependent homomeric complexes HEK293T cells transfected with non-targeting control or FANCD2 siRNA were co-transfected with HA-FANCI and GFP-FANCI constructs as indicated. Whole-cell extracts (WCE) were subjected to GFP immunoprecipitation followed by immunoblotting with the indicated antibodies. Extracts of HEK293T cells co-transfected with indicated combinations of HA-FANCI and GFP-FANCI constructs were subjected to GFP immunoprecipitation followed by immunoblotting with HA and GFP antibodies. U2OS cells were transfected with non-targeting control (CTRL) or UBL5 siRNA, subsequently transfected with a construct encoding DmrB-GFP-FANCI and then treated with 100 nM AP20187 (B/B homodimerizer) or left untreated. Cell extracts were analyzed by immunoblotting with the indicated antibodies.

    Techniques Used: Transfection, Construct, Immunoprecipitation

    UBL5 interacts with FANCI Whole-cell extracts (WCE) of HeLa cells transfected with Strep-HA-UBL5 plasmid were subjected to Strep-Tactin pull-down followed by immunoblotting with FANCI and HA antibodies. Whole-cell extracts (WCE) of HEK293T cells transfected with cDNA encoding GFP-FANCI were subjected to GFP immunoprecipitation followed by immunoblotting with UBL5 and GFP antibodies. Whole-cell extracts (WCE) of HeLa cells treated or not with 0.5 μM mitomycin C (MMC) for 24 h were subjected to immunoprecipitation with FANCI antibody or pre-immune serum (IgG) followed by immunoblotting with UBL5 and FANCI antibodies. Recombinant chicken FANCI (chFANCI) was incubated with His 6 -SUMO2 or His 6 -UBL5. Bound proteins were resolved by SDS–PAGE and visualized by colloidal blue staining. HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) were transfected with non-targeting or FANCD2 siRNAs for 48 h. Whole-cell extracts (WCE) were subjected to Strep-Tactin pull-down followed by immunoblotting with the indicated antibodies. * non-specific band. HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) were left untreated or synchronized by double thymidine block and released for the indicated times. Whole-cell extracts (WCE) were subjected to Strep-Tactin pull-down followed by immunoblotting with the indicated antibodies. * non-specific band.
    Figure Legend Snippet: UBL5 interacts with FANCI Whole-cell extracts (WCE) of HeLa cells transfected with Strep-HA-UBL5 plasmid were subjected to Strep-Tactin pull-down followed by immunoblotting with FANCI and HA antibodies. Whole-cell extracts (WCE) of HEK293T cells transfected with cDNA encoding GFP-FANCI were subjected to GFP immunoprecipitation followed by immunoblotting with UBL5 and GFP antibodies. Whole-cell extracts (WCE) of HeLa cells treated or not with 0.5 μM mitomycin C (MMC) for 24 h were subjected to immunoprecipitation with FANCI antibody or pre-immune serum (IgG) followed by immunoblotting with UBL5 and FANCI antibodies. Recombinant chicken FANCI (chFANCI) was incubated with His 6 -SUMO2 or His 6 -UBL5. Bound proteins were resolved by SDS–PAGE and visualized by colloidal blue staining. HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) were transfected with non-targeting or FANCD2 siRNAs for 48 h. Whole-cell extracts (WCE) were subjected to Strep-Tactin pull-down followed by immunoblotting with the indicated antibodies. * non-specific band. HeLa/Strep-HA-UBL5 cells induced or not with doxycycline (DOX) were left untreated or synchronized by double thymidine block and released for the indicated times. Whole-cell extracts (WCE) were subjected to Strep-Tactin pull-down followed by immunoblotting with the indicated antibodies. * non-specific band.

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Recombinant, Incubation, SDS Page, Staining, Blocking Assay

    Binding of UBL5 to FANCI facilitates formation of FANCI–FANCD2 heterodimers HeLa cells transfected with GFP-tagged FANCI WT or ∆UBL5 were harvested at the indicated times following addition of cycloheximide (CHX). Cell lysates were immunoblotted with GFP and β-tubulin antibodies. Quantification of GFP-FANCI levels in (A) by image analysis, normalized to β-tubulin levels. Mean values (± SD) from three independent experiments are shown. U2OS cells stably expressing HA-tagged FANCI WT or ∆UBL5 were treated or not with 1 μM mitomycin C (MMC) for 16 h. Cell extracts were analyzed by immunoblotting with HA and SMC1 antibodies. Extracts of HEK293T cells transfected with the indicated GFP-FANCI constructs were subjected to GFP immunoprecipitation followed by immunoblotting with FANCD2 and GFP antibodies. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 were fixed and incubated with antibodies against HA and FANCD2. The interaction between HA-FANCI and FANCD2 was visualized using PLA. Nuclei were stained with DAPI. Representative images are shown. Scale bar, 10 μm. Quantification of data in (E). Graph shows the quantification of PLA dots per nucleus. More than 200 cells were counted for each condition. Red lines indicate the mean of the data plotted. P -value was calculated by the Mann–Whitney U -test. Strep-GFP-FANCI WT or ∆UBL5 was purified from HEK293T cells and incubated with bacterially produced, recombinant His 6 -FANCD2. Complexes were immobilized on Strep-Tactin Sepharose and washed extensively. Bound material and input samples were then subjected to immunoblotting with His 6 and GFP antibodies.
    Figure Legend Snippet: Binding of UBL5 to FANCI facilitates formation of FANCI–FANCD2 heterodimers HeLa cells transfected with GFP-tagged FANCI WT or ∆UBL5 were harvested at the indicated times following addition of cycloheximide (CHX). Cell lysates were immunoblotted with GFP and β-tubulin antibodies. Quantification of GFP-FANCI levels in (A) by image analysis, normalized to β-tubulin levels. Mean values (± SD) from three independent experiments are shown. U2OS cells stably expressing HA-tagged FANCI WT or ∆UBL5 were treated or not with 1 μM mitomycin C (MMC) for 16 h. Cell extracts were analyzed by immunoblotting with HA and SMC1 antibodies. Extracts of HEK293T cells transfected with the indicated GFP-FANCI constructs were subjected to GFP immunoprecipitation followed by immunoblotting with FANCD2 and GFP antibodies. U2OS cells stably expressing HA-FANCI WT or ∆UBL5 were fixed and incubated with antibodies against HA and FANCD2. The interaction between HA-FANCI and FANCD2 was visualized using PLA. Nuclei were stained with DAPI. Representative images are shown. Scale bar, 10 μm. Quantification of data in (E). Graph shows the quantification of PLA dots per nucleus. More than 200 cells were counted for each condition. Red lines indicate the mean of the data plotted. P -value was calculated by the Mann–Whitney U -test. Strep-GFP-FANCI WT or ∆UBL5 was purified from HEK293T cells and incubated with bacterially produced, recombinant His 6 -FANCD2. Complexes were immobilized on Strep-Tactin Sepharose and washed extensively. Bound material and input samples were then subjected to immunoblotting with His 6 and GFP antibodies.

    Techniques Used: Binding Assay, Transfection, Stable Transfection, Expressing, Construct, Immunoprecipitation, Incubation, Proximity Ligation Assay, Staining, MANN-WHITNEY, Purification, Produced, Recombinant

    UBL5 has a direct role in promoting the FA pathway distinct from its pre-mRNA splicing involvement HeLa cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by immunoblotting with the indicated antibodies. * non-specific band. HeLa cells stably expressing HA-tagged FANCI were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by immunoblotting with the indicated antibodies. * non-specific band. Whole-cell extracts (WCE) of HeLa cells transfected with the indicated Strep-HA-UBL5 constructs were subjected to Strep-Tactin pull-down followed by immunoblotting with FANCI, SART1 and HA antibodies. U2OS cells stably expressing siRNA-resistant (siR) forms of indicated Strep-HA-UBL5 alleles were induced or not with doxycycline, transfected with non-targeting control (CTRL) or UBL5 siRNAs and treated with MMC for 24 h. Cells were then fixed and immunostained with FANCD2 antibody. FANCD2 foci formation in cells was enumerated, and cells containing more than five foci were defined as foci positive. More than 200 cells were counted for each condition. Results from three independent experiments (mean ± SD) are shown. Quantification of precocious sister chromatid separation in U2OS cells stably expressing doxycycline-inducible Strep-HA-UBL5(si R ) constructs after knockdown of endogenous UBL5. At least 200 metaphase spreads were counted in each experiment. Mean values (± SD) from three independent experiments are shown.
    Figure Legend Snippet: UBL5 has a direct role in promoting the FA pathway distinct from its pre-mRNA splicing involvement HeLa cells were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by immunoblotting with the indicated antibodies. * non-specific band. HeLa cells stably expressing HA-tagged FANCI were transfected with the indicated siRNAs for 48 h. Cell extracts were analyzed by immunoblotting with the indicated antibodies. * non-specific band. Whole-cell extracts (WCE) of HeLa cells transfected with the indicated Strep-HA-UBL5 constructs were subjected to Strep-Tactin pull-down followed by immunoblotting with FANCI, SART1 and HA antibodies. U2OS cells stably expressing siRNA-resistant (siR) forms of indicated Strep-HA-UBL5 alleles were induced or not with doxycycline, transfected with non-targeting control (CTRL) or UBL5 siRNAs and treated with MMC for 24 h. Cells were then fixed and immunostained with FANCD2 antibody. FANCD2 foci formation in cells was enumerated, and cells containing more than five foci were defined as foci positive. More than 200 cells were counted for each condition. Results from three independent experiments (mean ± SD) are shown. Quantification of precocious sister chromatid separation in U2OS cells stably expressing doxycycline-inducible Strep-HA-UBL5(si R ) constructs after knockdown of endogenous UBL5. At least 200 metaphase spreads were counted in each experiment. Mean values (± SD) from three independent experiments are shown.

    Techniques Used: Transfection, Stable Transfection, Expressing, Construct

    UBL5 is required for the integrity of the FA pathway Extracts of HeLa cells transfected with non-targeting control (CTRL) or UBL5 siRNAs for 48 h were analyzed by immunoblotting with the indicated antibodies. * non-specific band. HeLa cells stably expressing a doxycycline-inducible siRNA-resistant form (si R ) of Strep-HA-UBL5 were transfected with non-targeting control (CTRL) or UBL5 siRNAs for 48 h. Cell extracts were analyzed by immunoblotting with FANCI, FANCD2 and β-tubulin antibodies. HeLa cells transfected with non-targeting or UBL5 siRNAs were transfected with Strep-HA-UBL5 or siRNA-resistant form (si R ) of Strep-HA-UBL5. Cell extracts were analyzed by immunoblotting with HA and β-actin antibodies. HeLa cells transfected with non-targeting control (CTRL) or UBL5 siRNAs and then treated or not with MMC for 15 h were lysed and processed for immunoblotting with FANCI, FANCD2, UBL5 and β-tubulin antibodies. U2OS cells transfected with non-targeting control (CTRL) or UBL5 siRNAs were subjected to laser microirradiation in the presence of trioxsalen, fixed 2 h later and co-immunostained with FANCI, γ-H2AX and cyclin A antibodies. Representative images are shown. Scale bar, 10 μm. Clonogenic survival of U2OS cells transfected with indicated siRNAs and exposed to various doses of MMC. Results from at least three independent experiments (mean ± SEM) are shown. Clonogenic survival of U2OS/Strep-HA-UBL5(si R ) cells treated or not with doxycycline (DOX) to induce expression of the transgenes, transfected with the indicated siRNAs and then exposed to various doses of MMC. Results from three independent experiments (mean ± SEM) are shown.
    Figure Legend Snippet: UBL5 is required for the integrity of the FA pathway Extracts of HeLa cells transfected with non-targeting control (CTRL) or UBL5 siRNAs for 48 h were analyzed by immunoblotting with the indicated antibodies. * non-specific band. HeLa cells stably expressing a doxycycline-inducible siRNA-resistant form (si R ) of Strep-HA-UBL5 were transfected with non-targeting control (CTRL) or UBL5 siRNAs for 48 h. Cell extracts were analyzed by immunoblotting with FANCI, FANCD2 and β-tubulin antibodies. HeLa cells transfected with non-targeting or UBL5 siRNAs were transfected with Strep-HA-UBL5 or siRNA-resistant form (si R ) of Strep-HA-UBL5. Cell extracts were analyzed by immunoblotting with HA and β-actin antibodies. HeLa cells transfected with non-targeting control (CTRL) or UBL5 siRNAs and then treated or not with MMC for 15 h were lysed and processed for immunoblotting with FANCI, FANCD2, UBL5 and β-tubulin antibodies. U2OS cells transfected with non-targeting control (CTRL) or UBL5 siRNAs were subjected to laser microirradiation in the presence of trioxsalen, fixed 2 h later and co-immunostained with FANCI, γ-H2AX and cyclin A antibodies. Representative images are shown. Scale bar, 10 μm. Clonogenic survival of U2OS cells transfected with indicated siRNAs and exposed to various doses of MMC. Results from at least three independent experiments (mean ± SEM) are shown. Clonogenic survival of U2OS/Strep-HA-UBL5(si R ) cells treated or not with doxycycline (DOX) to induce expression of the transgenes, transfected with the indicated siRNAs and then exposed to various doses of MMC. Results from three independent experiments (mean ± SEM) are shown.

    Techniques Used: Transfection, Stable Transfection, Expressing

    2) Product Images from "Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells"

    Article Title: Bone morphogenetic protein 4 reduces global H3K4me3 to inhibit proliferation and promote differentiation of human neural stem cells

    Journal: bioRxiv

    doi: 10.1101/2020.01.22.915934

    BMP4 reduces global H3K4me3 promoter occupancy through reduction of its methyltransferases WDR82 and hSETD1A. A. Western blots shows WDR82 and human SETD1A/B expression following 100ng/ml BMP4 treatment. B. Representative images and quantitative graphs showing sphere formation in human StemPro® NSCs following treatment with siRNA for hSETD1A (siSETD1A) or shRNA for WDR82 (shWDR82) vectors versus controls (control siRNA, siCtrl and scrambled shRNA, scrCtrl). C and D. Real-time PCR (C) and western blots (D) showing expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN, following treatments as in B. E. Real-time PCR using DNA from ChIP with rabbit IgG and H3K4me3 and detected with promoter primers for OCT4, CCND1 and NESTIN following treatment with siSETD1A, shWDR82 or siCtrl, scrCtrl, in StemPro® NSCs. Error bars show the standard error of three independent experiments. (* p
    Figure Legend Snippet: BMP4 reduces global H3K4me3 promoter occupancy through reduction of its methyltransferases WDR82 and hSETD1A. A. Western blots shows WDR82 and human SETD1A/B expression following 100ng/ml BMP4 treatment. B. Representative images and quantitative graphs showing sphere formation in human StemPro® NSCs following treatment with siRNA for hSETD1A (siSETD1A) or shRNA for WDR82 (shWDR82) vectors versus controls (control siRNA, siCtrl and scrambled shRNA, scrCtrl). C and D. Real-time PCR (C) and western blots (D) showing expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN, following treatments as in B. E. Real-time PCR using DNA from ChIP with rabbit IgG and H3K4me3 and detected with promoter primers for OCT4, CCND1 and NESTIN following treatment with siSETD1A, shWDR82 or siCtrl, scrCtrl, in StemPro® NSCs. Error bars show the standard error of three independent experiments. (* p

    Techniques Used: Western Blot, Expressing, shRNA, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Ectopic expression of human SETD1A and WDR82 increases global H3K4me3 promoter occupancy in normal human astrocytes (HAs). A. Representative images and quantitative graphs show sphere formation from HAs following transfection with WDR82 (pcDNA3-WDR82) or human SETD1A (pET28-hSETD1A-MHL) expression plasmids, in comparison to controls (pcDNA3 and pET28- MHL). B and C. Real-time PCR (B) and western blots (C) show expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN. D. ChIP with rabbit IgG and H3K4me3 combined with real-time PCR using promoter primers for OCT4, CCND1 and NESTIN following HA transfection. Error bars show the standard deviation of three independent experiments. (* p
    Figure Legend Snippet: Ectopic expression of human SETD1A and WDR82 increases global H3K4me3 promoter occupancy in normal human astrocytes (HAs). A. Representative images and quantitative graphs show sphere formation from HAs following transfection with WDR82 (pcDNA3-WDR82) or human SETD1A (pET28-hSETD1A-MHL) expression plasmids, in comparison to controls (pcDNA3 and pET28- MHL). B and C. Real-time PCR (B) and western blots (C) show expression of hSETD1A, WDR82, OCT4, CCND1 and NESTIN. D. ChIP with rabbit IgG and H3K4me3 combined with real-time PCR using promoter primers for OCT4, CCND1 and NESTIN following HA transfection. Error bars show the standard deviation of three independent experiments. (* p

    Techniques Used: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Standard Deviation

    3) Product Images from "Targeting Somatostatin Receptors By Functionalized Mesoporous Silica Nanoparticles - Are We Striking Home?"

    Article Title: Targeting Somatostatin Receptors By Functionalized Mesoporous Silica Nanoparticles - Are We Striking Home?

    Journal: Nanotheranostics

    doi: 10.7150/ntno.23826

    SSTR2, 3 and 5 expression in cell lines employed in the study: indirect immunolabelling in a flow cytometry analysis. SSTR2, 3 and 5 immunolabelling results in paraformaldehyde (PFA)-fixed and saponin-permeabilized HEK293 and BON1 cells, along with matched control stains in viable non-permeabilized HEK293 cells are presented on panels A and B, respectively. As all the anti-SSTR antibodies (Abs) employed in the series on panel A target native epitopes within C -tails of the receptors (confined to cytoplasmic compartment), the cells were fixed with PFA and permeabilized with saponin before immunolabelling. Conversely, the immunolabelling of the cells on panel B involved primary Ab against distinct tags within extracellular N -termini of SSTRs, hence no permeabilization was required and the staining was done on viable non-permeabilized cells. Noteworthy, the pattern of signal from matched samples stained for the same target with Abs against its different epitopes (Abs to intracellular C -tails of receptors on panel A vs Abs to tags within extracellular domains of the same receptors on panel B) is almost identical, which signifies specificity of the data. Bimodal appearance of the populations on histograms, especially obvious in case of SSTR3- and 5-overexpressing cells, is explained by the oligoclonal nature of the cultures, with the resulting distribution being formed by progeny of two (or more) dominant clones. Staining for β-tubulin, a component of a cytoskeleton, on panels A and B was implemented as a positive or negative control of permeabilization, respectively. The cells were analyzed on LSRII cytometer; at least 15 000 of the gated events were captured. Every image represents an overlay histogram of two samples: black transparent charts stand for either non-stained controls or fully stained samples; shaded green charts reflect the corresponding secondary antibody - only stained controls. x-axis denotes sample emission [(505 nm longpass)/(530/30 nm bandpass)] upon stimulation with 488 nm laser; y-axis indicates the number of events registered. The data from a single representative experiment (performed in duplicate) is shown; the complete series has been independently performed at least three times.
    Figure Legend Snippet: SSTR2, 3 and 5 expression in cell lines employed in the study: indirect immunolabelling in a flow cytometry analysis. SSTR2, 3 and 5 immunolabelling results in paraformaldehyde (PFA)-fixed and saponin-permeabilized HEK293 and BON1 cells, along with matched control stains in viable non-permeabilized HEK293 cells are presented on panels A and B, respectively. As all the anti-SSTR antibodies (Abs) employed in the series on panel A target native epitopes within C -tails of the receptors (confined to cytoplasmic compartment), the cells were fixed with PFA and permeabilized with saponin before immunolabelling. Conversely, the immunolabelling of the cells on panel B involved primary Ab against distinct tags within extracellular N -termini of SSTRs, hence no permeabilization was required and the staining was done on viable non-permeabilized cells. Noteworthy, the pattern of signal from matched samples stained for the same target with Abs against its different epitopes (Abs to intracellular C -tails of receptors on panel A vs Abs to tags within extracellular domains of the same receptors on panel B) is almost identical, which signifies specificity of the data. Bimodal appearance of the populations on histograms, especially obvious in case of SSTR3- and 5-overexpressing cells, is explained by the oligoclonal nature of the cultures, with the resulting distribution being formed by progeny of two (or more) dominant clones. Staining for β-tubulin, a component of a cytoskeleton, on panels A and B was implemented as a positive or negative control of permeabilization, respectively. The cells were analyzed on LSRII cytometer; at least 15 000 of the gated events were captured. Every image represents an overlay histogram of two samples: black transparent charts stand for either non-stained controls or fully stained samples; shaded green charts reflect the corresponding secondary antibody - only stained controls. x-axis denotes sample emission [(505 nm longpass)/(530/30 nm bandpass)] upon stimulation with 488 nm laser; y-axis indicates the number of events registered. The data from a single representative experiment (performed in duplicate) is shown; the complete series has been independently performed at least three times.

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining, Clone Assay, Negative Control

    4) Product Images from "Interferon-Inducible Protein IFIX?1 Functions as a Negative Regulator of HDM2"

    Article Title: Interferon-Inducible Protein IFIX?1 Functions as a Negative Regulator of HDM2

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.26.5.1979-1996.2006

    IFIXα1 interacts with HDM2. (A) HDM2 interacts with IFIXα1 and IFIXβ1. 293T cells were cotransfected with HDM2 (2.5 μg) and EGFP vector (vector) (2.5 μg), EGFP-tagged IFIXα1 (α1) (2.5 μg), or IFIXβ1 (β1) (2.5 μg). Forty-eight hours posttransfection, cell lysates (500 μg) were immunoprecipitated with an anti-HDM2 antibody, and Western blotting was performed using an anti-GFP or anti-HDM2 antibody. (B) A reciprocal experiment that used anti-GFP antibody for IP and Western blotting with anti-IFIX or anti-HDM2 antibodies. (C) IFIXα1 interacts with HDM2 in the IFIXα1 stable cell lines. Cell lysates (600 μg) isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) or the empty vector cells (V) were immunoprecipitated using anti-HDM2 antibody and analyzed by Western blotting with anti-IFIXα or anti-HDM2 antibody. (D) The HDM2(1-441) mutant interacts with IFIXα1. 293T cells were transfected with HDM2 or the HDM2(1-441) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (V), followed by IP with anti-HDM2 antibody and Western blotting with either anti-HDM2 or anti-GFP antibody. (E and F) Amino acid region 150 to 230 of HDM2 interacts with IFIXα1. 293T cells were cotransfected with HDM2 or the HDM2(Δ150-230) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (E) or FLAG-IFIXα1 (α1) or FLAG empty vector (F), followed by IP/Western blotting with the indicated antibodies. An arrowhead indicates the HDM2 band (F). The untransfected 293T cells served as controls (C). (G) A summary of IFIXα1 binding by HDM2 and the HDM2(Δ150-230) and HDM2(1-441) mutants. (H and I) The HIN domain of IFIXα1 interacts with HDM2. (H) 293T cells transfected with HDM2 and EGFP empty vector, EGFP-IFIXα1, EGFP-IFIX-N (N), or EGFP-IFIX-HIN (HIN), followed by IP with anti-GFP antibody (left panel) or anti-HDM2 antibody (right panel) and Western blotting with anti-HDM2 or anti-GFP antibody. Untransfected 293T cells served as controls. (I) A summary of HDM2 binding by IFIXα1, IFIXβ1, IFIX-N, and IFIX-HIN.
    Figure Legend Snippet: IFIXα1 interacts with HDM2. (A) HDM2 interacts with IFIXα1 and IFIXβ1. 293T cells were cotransfected with HDM2 (2.5 μg) and EGFP vector (vector) (2.5 μg), EGFP-tagged IFIXα1 (α1) (2.5 μg), or IFIXβ1 (β1) (2.5 μg). Forty-eight hours posttransfection, cell lysates (500 μg) were immunoprecipitated with an anti-HDM2 antibody, and Western blotting was performed using an anti-GFP or anti-HDM2 antibody. (B) A reciprocal experiment that used anti-GFP antibody for IP and Western blotting with anti-IFIX or anti-HDM2 antibodies. (C) IFIXα1 interacts with HDM2 in the IFIXα1 stable cell lines. Cell lysates (600 μg) isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) or the empty vector cells (V) were immunoprecipitated using anti-HDM2 antibody and analyzed by Western blotting with anti-IFIXα or anti-HDM2 antibody. (D) The HDM2(1-441) mutant interacts with IFIXα1. 293T cells were transfected with HDM2 or the HDM2(1-441) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (V), followed by IP with anti-HDM2 antibody and Western blotting with either anti-HDM2 or anti-GFP antibody. (E and F) Amino acid region 150 to 230 of HDM2 interacts with IFIXα1. 293T cells were cotransfected with HDM2 or the HDM2(Δ150-230) mutant and EGFP-IFIXα1 (α1) or EGFP empty vector (E) or FLAG-IFIXα1 (α1) or FLAG empty vector (F), followed by IP/Western blotting with the indicated antibodies. An arrowhead indicates the HDM2 band (F). The untransfected 293T cells served as controls (C). (G) A summary of IFIXα1 binding by HDM2 and the HDM2(Δ150-230) and HDM2(1-441) mutants. (H and I) The HIN domain of IFIXα1 interacts with HDM2. (H) 293T cells transfected with HDM2 and EGFP empty vector, EGFP-IFIXα1, EGFP-IFIX-N (N), or EGFP-IFIX-HIN (HIN), followed by IP with anti-GFP antibody (left panel) or anti-HDM2 antibody (right panel) and Western blotting with anti-HDM2 or anti-GFP antibody. Untransfected 293T cells served as controls. (I) A summary of HDM2 binding by IFIXα1, IFIXβ1, IFIX-N, and IFIX-HIN.

    Techniques Used: Plasmid Preparation, Immunoprecipitation, Western Blot, Stable Transfection, Isolation, Mutagenesis, Transfection, Binding Assay

    IFIXα1 mediates the IFN-α-induced HDM2 downregulation. (A) IFN-α treatment reduces the HDM2 protein levels. Raji cells were treated with or without IFN-α (2,000 U/ml) for the indicated times (0, 24, 48, and 72 h), followed by Western blotting using the antibodies against HDM2, IFIXα, PKR, and α-tubulin. (B) IFN-α induces the expression of both IFIXα1 and IFI16 proteins. Raji cells were treated with or without IFN-α (2,000 U/ml) for 72 h, followed by Western blotting using the antibodies against HDM2, IFI16, IFIXα, and α-tubulin. (C) IFIX siRNA transfection reverses the IFN-α-mediated downregulation of HDM2. Raji cells growing in 0.2% FCS DMEM/F12 medium with or without IFN-α treatment (2,000 U/ml) for 72 h were analyzed by Western blotting using antibodies against HDM2, IFIXα, IFI16, and α-tubulin (left panel). The protein expression was likewise analyzed in the Raji cells transfected with either IFIX siRNA (100 nM) or the NS siRNA (100 nM) in 0.2% FCS DMEM/F12 medium, followed by IFN-α treatment (2,000 U/ml) for 72 h (right panel). (D) IFN-α treatment increased the IFIXα and HDM2 interaction. Cell lysates (800 μg) isolated from Raji cells treated with (+) or without (−) IFN-α (2,000 U/ml) for 48 h, followed by IP with anti-HDM2 or immunoglobulin G antibody and Western blotting with anti-HDM2 and anti-IFIXα antibodies.
    Figure Legend Snippet: IFIXα1 mediates the IFN-α-induced HDM2 downregulation. (A) IFN-α treatment reduces the HDM2 protein levels. Raji cells were treated with or without IFN-α (2,000 U/ml) for the indicated times (0, 24, 48, and 72 h), followed by Western blotting using the antibodies against HDM2, IFIXα, PKR, and α-tubulin. (B) IFN-α induces the expression of both IFIXα1 and IFI16 proteins. Raji cells were treated with or without IFN-α (2,000 U/ml) for 72 h, followed by Western blotting using the antibodies against HDM2, IFI16, IFIXα, and α-tubulin. (C) IFIX siRNA transfection reverses the IFN-α-mediated downregulation of HDM2. Raji cells growing in 0.2% FCS DMEM/F12 medium with or without IFN-α treatment (2,000 U/ml) for 72 h were analyzed by Western blotting using antibodies against HDM2, IFIXα, IFI16, and α-tubulin (left panel). The protein expression was likewise analyzed in the Raji cells transfected with either IFIX siRNA (100 nM) or the NS siRNA (100 nM) in 0.2% FCS DMEM/F12 medium, followed by IFN-α treatment (2,000 U/ml) for 72 h (right panel). (D) IFN-α treatment increased the IFIXα and HDM2 interaction. Cell lysates (800 μg) isolated from Raji cells treated with (+) or without (−) IFN-α (2,000 U/ml) for 48 h, followed by IP with anti-HDM2 or immunoglobulin G antibody and Western blotting with anti-HDM2 and anti-IFIXα antibodies.

    Techniques Used: Western Blot, Expressing, Transfection, Isolation

    IFIXα1 destabilizes HDM2. (A) Inverse relationship between IFIXα1 and HDM2 expression. Cell lysates isolated from IFIXα1 stable MDA-MB-468 cell lines (X-1 and X-2) and the vector control cell lines (V) were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (B) IFIXα1 is responsible for HDM2 downregulation. MDA-MB-468 (X-1) cells were transfected with IFIX siRNA (100 nM) or NS siRNA (100 nM). Forty-eight hours after transfection, the expression levels of HDM2, IFIXα1, p53, and α-tubulin were analyzed by Western blotting. (C) IFIXα1 has little effect on the steady-state mRNA levels of HDM2 in MDA-MB-468 cells. Total RNA (10 μg) isolated from the parental MDA-MB-468 (C), the IFIX stable cell lines (X-1 and X-2), and the empty vector transfected cells (V) was analyzed by Northern blotting using HDM2, p53, or IFIXα1 cDNA as a probe. The 18S and 28S rRNAs are shown as loading controls. (D) IFIXα1 destabilizes HDM2. H1299 cells were cotransfected with HDM2 (0.7 μg) and the empty vector (V) (1.3 μg) or FLAG-IFIXα1 (IFIXα1) (1.3 μg). Twenty-four hours posttransfection, cells were treated with CHX (100 μg/ml). Cell lysates were isolated at 0, 15, and 30 min after CHX treatment for Western blotting using antibodies against HDM2, IFIXα, and α-tubulin. A representative experiment is shown. (E) The amount of HDM2 protein at zero time point was arbitrarily set at 100%. The percentage of HDM2 protein remaining was determined using Bio-Rad software. The results obtained from three independent experiments are shown.
    Figure Legend Snippet: IFIXα1 destabilizes HDM2. (A) Inverse relationship between IFIXα1 and HDM2 expression. Cell lysates isolated from IFIXα1 stable MDA-MB-468 cell lines (X-1 and X-2) and the vector control cell lines (V) were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (B) IFIXα1 is responsible for HDM2 downregulation. MDA-MB-468 (X-1) cells were transfected with IFIX siRNA (100 nM) or NS siRNA (100 nM). Forty-eight hours after transfection, the expression levels of HDM2, IFIXα1, p53, and α-tubulin were analyzed by Western blotting. (C) IFIXα1 has little effect on the steady-state mRNA levels of HDM2 in MDA-MB-468 cells. Total RNA (10 μg) isolated from the parental MDA-MB-468 (C), the IFIX stable cell lines (X-1 and X-2), and the empty vector transfected cells (V) was analyzed by Northern blotting using HDM2, p53, or IFIXα1 cDNA as a probe. The 18S and 28S rRNAs are shown as loading controls. (D) IFIXα1 destabilizes HDM2. H1299 cells were cotransfected with HDM2 (0.7 μg) and the empty vector (V) (1.3 μg) or FLAG-IFIXα1 (IFIXα1) (1.3 μg). Twenty-four hours posttransfection, cells were treated with CHX (100 μg/ml). Cell lysates were isolated at 0, 15, and 30 min after CHX treatment for Western blotting using antibodies against HDM2, IFIXα, and α-tubulin. A representative experiment is shown. (E) The amount of HDM2 protein at zero time point was arbitrarily set at 100%. The percentage of HDM2 protein remaining was determined using Bio-Rad software. The results obtained from three independent experiments are shown.

    Techniques Used: Expressing, Isolation, Multiple Displacement Amplification, Plasmid Preparation, Western Blot, Transfection, Stable Transfection, Northern Blot, Software

    IFIXα1 stabilizes p53 protein. (A) IFIXα1 exerts different effects on the p53 and HDM2 levels in p53-expressing cells. Total cell lysates isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) and the vector control (V) cell lines were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (B) The p53 status influences the IFIXα1 effect on HDM2 levels. HCT116 and HCT116(p53 −/− ) cells were transfected with EGFP vector or EGFP-IFIXα1. Forty-eight hours after transfection, the GFP-positive cells were collected using FACS. Cell lysates were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (C) IFIXα1 induces the steady-state HDM2 mRNA level but has little effect on p53 mRNA levels in MCF-7 cells. Total RNA (10 μg) isolated from the parental MCF-7 (C) and the stable cell lines transfected with the empty vector (V) or IFIXα1 expression vector (X-1 and X-2) was analyzed by Northern blotting using HDM2, p53, or IFIXα1 cDNA as a probe. The 18S and 28S rRNAs are shown as loading controls. (D) IFIXα1 increases p53 protein stability. The IFIXα1 stable MCF-7 (X-1 and X-2) and the vector control (V) cells were treated with CHX (100 μg /ml) for the time indicated. Cell lysates were analyzed for the expression of p53 and α-tubulin. (E and F) Depletion of IFIXα1 reduces p53 and p21 CIP1 expression levels. The IFIXα1 stable MCF-7 cell line, X-1, was transfected with siRNA specific to IFIXα (IFIX) (100 nM) or NS siRNA (100 nM). Forty-eight hours after transfection, the expression levels of p53, IFIXα1, p21 CIP1 , and α-tubulin were analyzed by Western blotting.
    Figure Legend Snippet: IFIXα1 stabilizes p53 protein. (A) IFIXα1 exerts different effects on the p53 and HDM2 levels in p53-expressing cells. Total cell lysates isolated from the IFIXα1 stable MCF-7 cell lines (X-1 and X-2) and the vector control (V) cell lines were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (B) The p53 status influences the IFIXα1 effect on HDM2 levels. HCT116 and HCT116(p53 −/− ) cells were transfected with EGFP vector or EGFP-IFIXα1. Forty-eight hours after transfection, the GFP-positive cells were collected using FACS. Cell lysates were analyzed by Western blotting using antibodies against HDM2, p53, IFIXα, and α-tubulin. (C) IFIXα1 induces the steady-state HDM2 mRNA level but has little effect on p53 mRNA levels in MCF-7 cells. Total RNA (10 μg) isolated from the parental MCF-7 (C) and the stable cell lines transfected with the empty vector (V) or IFIXα1 expression vector (X-1 and X-2) was analyzed by Northern blotting using HDM2, p53, or IFIXα1 cDNA as a probe. The 18S and 28S rRNAs are shown as loading controls. (D) IFIXα1 increases p53 protein stability. The IFIXα1 stable MCF-7 (X-1 and X-2) and the vector control (V) cells were treated with CHX (100 μg /ml) for the time indicated. Cell lysates were analyzed for the expression of p53 and α-tubulin. (E and F) Depletion of IFIXα1 reduces p53 and p21 CIP1 expression levels. The IFIXα1 stable MCF-7 cell line, X-1, was transfected with siRNA specific to IFIXα (IFIX) (100 nM) or NS siRNA (100 nM). Forty-eight hours after transfection, the expression levels of p53, IFIXα1, p21 CIP1 , and α-tubulin were analyzed by Western blotting.

    Techniques Used: Expressing, Isolation, Plasmid Preparation, Western Blot, Transfection, FACS, Stable Transfection, Northern Blot

    IFIX-HIN is sufficient to downregulate HDM2. (A) IFIX-HIN downregulates HDM2 expression. 293T cells were transfected with EGFP empty vector (EGFP) or EGFP-IFIX-HIN. MG132 (10 μM) treatment started at 5 h before harvest. Cell lysates isolated from the GFP-positive cells were analyzed by Western blotting using antibodies against HDM2, EGFP, and α-tubulin. (B to D) IFIX-HIN induces p53 and p21 CIP1 . H1299 cells were transfected with p53 (0.1 μg) and increasing amounts (0.5, 1.0, and 1.8 μg) of the FLAG-tagged IFIXα1 (B), IFIX-HIN (C), or IFIX-N (D), followed by Western blotting using antibodies against p53, IFIXα (B), FLAG (C and D), p21 CIP1 , and α-tubulin at 24 h posttransfection. (E) IFIXα1 induces p21 CIP1 mRNA expression. H1299 cells were cotransfected with p53 (0.5 μg) and 5.5 μg of FLAG-tagged empty vector (V), IFIXα1 (α1), IFIX-HIN (HIN), or IFIX-N (N). At 24 h posttransfection, total RNA (10 μg) isolated from these cells was analyzed by Northern blotting using p21 CIP1 or IFIX cDNA as a probe. The 18S and 28S rRNAs served as loading controls.
    Figure Legend Snippet: IFIX-HIN is sufficient to downregulate HDM2. (A) IFIX-HIN downregulates HDM2 expression. 293T cells were transfected with EGFP empty vector (EGFP) or EGFP-IFIX-HIN. MG132 (10 μM) treatment started at 5 h before harvest. Cell lysates isolated from the GFP-positive cells were analyzed by Western blotting using antibodies against HDM2, EGFP, and α-tubulin. (B to D) IFIX-HIN induces p53 and p21 CIP1 . H1299 cells were transfected with p53 (0.1 μg) and increasing amounts (0.5, 1.0, and 1.8 μg) of the FLAG-tagged IFIXα1 (B), IFIX-HIN (C), or IFIX-N (D), followed by Western blotting using antibodies against p53, IFIXα (B), FLAG (C and D), p21 CIP1 , and α-tubulin at 24 h posttransfection. (E) IFIXα1 induces p21 CIP1 mRNA expression. H1299 cells were cotransfected with p53 (0.5 μg) and 5.5 μg of FLAG-tagged empty vector (V), IFIXα1 (α1), IFIX-HIN (HIN), or IFIX-N (N). At 24 h posttransfection, total RNA (10 μg) isolated from these cells was analyzed by Northern blotting using p21 CIP1 or IFIX cDNA as a probe. The 18S and 28S rRNAs served as loading controls.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Isolation, Western Blot, Northern Blot

    5) Product Images from "Gemin5 promotes IRES interaction and translation control through its C-terminal region"

    Article Title: Gemin5 promotes IRES interaction and translation control through its C-terminal region

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1212

    Gemin5 binds directly to domain 5 of the FMDV IRES. ( A ) Secondary structure of the FMDV IRES with indication of structural domains. Thin lines at the bottom depict the approximate location of transcripts (IRES, 1-2, 3, 4-5, 5, 5-H and 5-ss) used in this work. ( B ) UV-crosslinking assay of BHK-21 S10 cell extracts with radiolabelled IRES, or domain 1-2, 3, 4-5 and 5 RNAs. Arrows indicate the position of p220, p170, p120 and p80 photo-crosslinked products. ( C ) UV-crosslinking assay of radiolabelled domain 5, domain 5 hairpin (5-H) and the single-stranded region of domain (5-ss). ( D ) Immunoprecipitation of Gemin5 from BHK-21 cell extracts photo-crosslinked to radiolabelled transcripts IRES, 1-2, 5, 5-H and 5-ss. Samples were fractionated on 8% SDS-PAGE. In all cases the input (In) corresponds to 5% of the immunoprecipitation (IP) sample. ( E ) Effect of destabilization of the hairpin of domain 5 in protein photocrosslinking. Secondary structure of wild type (wt) domain 5 (corresponding to probe 5 in Figures 1 B–D); substitutions designed to destabilize the hairpin in mutants 5-stem or 5-base, and to restore the RNA structure in mutant 5-rest are marked with a rectangle (top panel). Autoradiograph of a UV-crosslinking assay carried out with the indicated 32 P-labelled mutant RNAs and BHK-21 S10 extracts, fractionated on SDS-PAGE 8% (bottom panel). Thin lines point to p170, p120 and p80. ( F ). Effect of the primary sequence composition within the 5′ and 3′ region of the hairpin in protein photocrosslinking. Substitutions designed to modify the primary sequence of the 5′ (mutants 5-sm, 5-scr) and 3′ sequence (5-U/A) of domain 5 (top panel) are marked with a rectangle. UV-crosslinking assay of the indicated 32 P-labelled mutant RNAs and BHK-21 S10 extracts (bottom panel).
    Figure Legend Snippet: Gemin5 binds directly to domain 5 of the FMDV IRES. ( A ) Secondary structure of the FMDV IRES with indication of structural domains. Thin lines at the bottom depict the approximate location of transcripts (IRES, 1-2, 3, 4-5, 5, 5-H and 5-ss) used in this work. ( B ) UV-crosslinking assay of BHK-21 S10 cell extracts with radiolabelled IRES, or domain 1-2, 3, 4-5 and 5 RNAs. Arrows indicate the position of p220, p170, p120 and p80 photo-crosslinked products. ( C ) UV-crosslinking assay of radiolabelled domain 5, domain 5 hairpin (5-H) and the single-stranded region of domain (5-ss). ( D ) Immunoprecipitation of Gemin5 from BHK-21 cell extracts photo-crosslinked to radiolabelled transcripts IRES, 1-2, 5, 5-H and 5-ss. Samples were fractionated on 8% SDS-PAGE. In all cases the input (In) corresponds to 5% of the immunoprecipitation (IP) sample. ( E ) Effect of destabilization of the hairpin of domain 5 in protein photocrosslinking. Secondary structure of wild type (wt) domain 5 (corresponding to probe 5 in Figures 1 B–D); substitutions designed to destabilize the hairpin in mutants 5-stem or 5-base, and to restore the RNA structure in mutant 5-rest are marked with a rectangle (top panel). Autoradiograph of a UV-crosslinking assay carried out with the indicated 32 P-labelled mutant RNAs and BHK-21 S10 extracts, fractionated on SDS-PAGE 8% (bottom panel). Thin lines point to p170, p120 and p80. ( F ). Effect of the primary sequence composition within the 5′ and 3′ region of the hairpin in protein photocrosslinking. Substitutions designed to modify the primary sequence of the 5′ (mutants 5-sm, 5-scr) and 3′ sequence (5-U/A) of domain 5 (top panel) are marked with a rectangle. UV-crosslinking assay of the indicated 32 P-labelled mutant RNAs and BHK-21 S10 extracts (bottom panel).

    Techniques Used: Immunoprecipitation, SDS Page, Mutagenesis, Autoradiography, Sequencing

    Impact of Gemin5 on IRES SHAPE reactivity. ( A ) RNA SHAPE reactivity as a function of nucleotide position for the free RNA treated with NMIA is depicted at the top panel. SHAPE reactivity values observed on addition of purified Gemin5 (15, 75 nM) are shown (top to bottom panels). Bars are coloured according to reactivity. Numbers depict nucleotides showing larger differences in SHAPE reactivity. ( B ) SHAPE difference plots of the IRES RNA incubated with Gemin5 relative to free RNA. Arrows below the bottom panel indicate the border of IRES domains 2, 3, 4 and 5.
    Figure Legend Snippet: Impact of Gemin5 on IRES SHAPE reactivity. ( A ) RNA SHAPE reactivity as a function of nucleotide position for the free RNA treated with NMIA is depicted at the top panel. SHAPE reactivity values observed on addition of purified Gemin5 (15, 75 nM) are shown (top to bottom panels). Bars are coloured according to reactivity. Numbers depict nucleotides showing larger differences in SHAPE reactivity. ( B ) SHAPE difference plots of the IRES RNA incubated with Gemin5 relative to free RNA. Arrows below the bottom panel indicate the border of IRES domains 2, 3, 4 and 5.

    Techniques Used: Purification, Incubation

    Modification of the difference in SHAPE reactivity induced upon addition of Gemin5 and PTB. ( A ) SHAPE difference profiles of the IRES incubated with Gemin5 (75 nM), PTB (900 nM), alone or combined, relative to free RNA. The RNA region where most of the rearrangements are observed upon addition of the proteins is highlighted by a dotted rectangle. ( B ) Schematic representation of the Gemin5 (yellow) and PTB (blue) individual binding sites and the conformational reorganization of domain 5 (highlighted with arrows) induced by the combined addition of both proteins (grey).
    Figure Legend Snippet: Modification of the difference in SHAPE reactivity induced upon addition of Gemin5 and PTB. ( A ) SHAPE difference profiles of the IRES incubated with Gemin5 (75 nM), PTB (900 nM), alone or combined, relative to free RNA. The RNA region where most of the rearrangements are observed upon addition of the proteins is highlighted by a dotted rectangle. ( B ) Schematic representation of the Gemin5 (yellow) and PTB (blue) individual binding sites and the conformational reorganization of domain 5 (highlighted with arrows) induced by the combined addition of both proteins (grey).

    Techniques Used: Modification, Incubation, Binding Assay

    The C-terminal region of Gemin5 represses translation. ( A ) Increasing amounts of RNA expressing G5-13WD were added to RRL prior to addition of a bicistronic RNA (200 ng) bearing the FMDV IRES in the intercistronic region. 35 S-labelled proteins were resolved in 12% SDS-PAGE, and the intensity of 35 S-labelled luciferase (squares) (IRES-dependent translation) and CAT (triangles) (5′-end dependent translation) proteins was measured in a densitometer. ( B ) Increasing amounts of an RNA expressing the C-terminal region of Gemin5 (G5-Cter) were added to RRL prior to addition of a bicistronic RNA. Values correspond to the mean (±SD) of three assays.
    Figure Legend Snippet: The C-terminal region of Gemin5 represses translation. ( A ) Increasing amounts of RNA expressing G5-13WD were added to RRL prior to addition of a bicistronic RNA (200 ng) bearing the FMDV IRES in the intercistronic region. 35 S-labelled proteins were resolved in 12% SDS-PAGE, and the intensity of 35 S-labelled luciferase (squares) (IRES-dependent translation) and CAT (triangles) (5′-end dependent translation) proteins was measured in a densitometer. ( B ) Increasing amounts of an RNA expressing the C-terminal region of Gemin5 (G5-Cter) were added to RRL prior to addition of a bicistronic RNA. Values correspond to the mean (±SD) of three assays.

    Techniques Used: Expressing, SDS Page, Luciferase

    Identification of the Gemin5 region involved in IRES interaction. ( A ) Diagram of the His-tagged Gemin5 proteins used in this study. Numbers indicate the amino acids encompassed by Gemin5 (full-length protein), G5-13WD (N-terminal region) or G5-Cter (C-terminal region). The Xpress epitope at the N-terminal end present in all proteins and the epitope recognized by anti-Gemin5 antibody are depicted by a grey or white diamonds, respectively; grey ovals depict the 13 WD repeats. ( B ). Schematic representation of the RNA-binding assay (top). Dotted circles depict agarose beads bound to anti-Xpress antibody; Xpress-tagged Gemin5 is depicted by grey ovals with a grey diamond, radiolabelled RNA is depicted in black. Autoradiograph of a denaturing 6% acrylamide gel, 7 M Urea loaded with RNAs isolated from protein G-Xpress antibody beads coupled to the indicated proteins (bottom). ( C ) UV-crosslinking (UV-XL) assay conducted with increasing amounts (0 to 200 ng) of purified His-tagged G5-Cter and radiolabelled domain 5. The mobility of the same protein detected by western blot (WB) using anti-Gemin5 is shown on the right.
    Figure Legend Snippet: Identification of the Gemin5 region involved in IRES interaction. ( A ) Diagram of the His-tagged Gemin5 proteins used in this study. Numbers indicate the amino acids encompassed by Gemin5 (full-length protein), G5-13WD (N-terminal region) or G5-Cter (C-terminal region). The Xpress epitope at the N-terminal end present in all proteins and the epitope recognized by anti-Gemin5 antibody are depicted by a grey or white diamonds, respectively; grey ovals depict the 13 WD repeats. ( B ). Schematic representation of the RNA-binding assay (top). Dotted circles depict agarose beads bound to anti-Xpress antibody; Xpress-tagged Gemin5 is depicted by grey ovals with a grey diamond, radiolabelled RNA is depicted in black. Autoradiograph of a denaturing 6% acrylamide gel, 7 M Urea loaded with RNAs isolated from protein G-Xpress antibody beads coupled to the indicated proteins (bottom). ( C ) UV-crosslinking (UV-XL) assay conducted with increasing amounts (0 to 200 ng) of purified His-tagged G5-Cter and radiolabelled domain 5. The mobility of the same protein detected by western blot (WB) using anti-Gemin5 is shown on the right.

    Techniques Used: RNA Binding Assay, Autoradiography, Acrylamide Gel Assay, Isolation, Purification, Western Blot

    Summary of SHAPE reactivity of the IRES element. Nucleotide SHAPE reactivity observed in the free RNA is represented by a coloured scale ( > 10, grey, > 25, yellow; > 50, orange). The nucleotides protected from RNA attack in SHAPE reactivity assays upon addition of Gemin5, or PTB, are depicted by yellow diamonds and blue rectangles, respectively.
    Figure Legend Snippet: Summary of SHAPE reactivity of the IRES element. Nucleotide SHAPE reactivity observed in the free RNA is represented by a coloured scale ( > 10, grey, > 25, yellow; > 50, orange). The nucleotides protected from RNA attack in SHAPE reactivity assays upon addition of Gemin5, or PTB, are depicted by yellow diamonds and blue rectangles, respectively.

    Techniques Used:

    6) Product Images from "Cyclin C is a haploinsufficient tumor suppressor"

    Article Title: Cyclin C is a haploinsufficient tumor suppressor

    Journal: Nature cell biology

    doi: 10.1038/ncb3046

    Gene expression analyses of cyclin C-null cells. ( a ) CDK8 was immunoprecipitated (IP) from wild-type (WT) or cyclin C Δ/Δ (C-KO) MEFs and used for in vitro kinase reactions with recombinant carboxy terminal domain (CTD) of RNA polymerase II as a substrate, in the presence of γ[ 32 P]ATP. C-KO + cyclin C: cyclin C Δ/Δ MEFs engineered to ectopically express cyclin C. Note that re-expression of cyclin C restored CDK8 kinase activity in cyclin C Δ/Δ cells. IgG was used for control immunoprecipitation. 32 P-CTD denotes phosphorylated CTD, detected by autoradiography. Lower panel, CTD was visualized by Coomassie staining. ( b ) Lysates from wild-type (WT), cyclin C +/Δ (C-HET) and cyclin C Δ/Δ (C-KO) ES cells were probed with an antibody against phospho-Ser 2 and phospho-Ser 5 of RNA polymerase II CTD. Lower panel: immunoblotting with an anti-CTD antibody. ( c ) Cyclin C, CDK8, MED12 or MED13 were immunoprecipitated (IP) from wild-type or C-KO MEFs; the immunoblots were probed with the indicated antibodies, along with whole cell extracts (Input). ( d ) Scatterplot showing log of the normalized gene expression in control (x-axis) vs. C Δ/Δ (y-axis) MEFs, ES cells (ESC) and E18.5 brains. Parallel lines indicate 2-fold change (up- or down-regulation) in transcript levels. ( e ) Scatterplot showing log of the normalized gene expression values in control brains (x-axis) vs. control ES cells (y-axis). This is a control to panel d . It illustrates that different compartments (brain, ES cells) display very distinct gene expression patterns, as expected.
    Figure Legend Snippet: Gene expression analyses of cyclin C-null cells. ( a ) CDK8 was immunoprecipitated (IP) from wild-type (WT) or cyclin C Δ/Δ (C-KO) MEFs and used for in vitro kinase reactions with recombinant carboxy terminal domain (CTD) of RNA polymerase II as a substrate, in the presence of γ[ 32 P]ATP. C-KO + cyclin C: cyclin C Δ/Δ MEFs engineered to ectopically express cyclin C. Note that re-expression of cyclin C restored CDK8 kinase activity in cyclin C Δ/Δ cells. IgG was used for control immunoprecipitation. 32 P-CTD denotes phosphorylated CTD, detected by autoradiography. Lower panel, CTD was visualized by Coomassie staining. ( b ) Lysates from wild-type (WT), cyclin C +/Δ (C-HET) and cyclin C Δ/Δ (C-KO) ES cells were probed with an antibody against phospho-Ser 2 and phospho-Ser 5 of RNA polymerase II CTD. Lower panel: immunoblotting with an anti-CTD antibody. ( c ) Cyclin C, CDK8, MED12 or MED13 were immunoprecipitated (IP) from wild-type or C-KO MEFs; the immunoblots were probed with the indicated antibodies, along with whole cell extracts (Input). ( d ) Scatterplot showing log of the normalized gene expression in control (x-axis) vs. C Δ/Δ (y-axis) MEFs, ES cells (ESC) and E18.5 brains. Parallel lines indicate 2-fold change (up- or down-regulation) in transcript levels. ( e ) Scatterplot showing log of the normalized gene expression values in control brains (x-axis) vs. control ES cells (y-axis). This is a control to panel d . It illustrates that different compartments (brain, ES cells) display very distinct gene expression patterns, as expected.

    Techniques Used: Expressing, Immunoprecipitation, In Vitro, Recombinant, Activity Assay, Autoradiography, Staining, Western Blot

    7) Product Images from "A novel P53/ POMC/Gαs/ SASH1 autoregulatory feedback loop activates mutated SASH1 to cause pathologic hyperpigmentation"

    Article Title: A novel P53/ POMC/Gαs/ SASH1 autoregulatory feedback loop activates mutated SASH1 to cause pathologic hyperpigmentation

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13022

    Reciprocal induction between p53 and SASH 1 is induced in normal cells. ( A ) and ( B ) Exogenous p53 ( HA ‐p53) triggers exogenous SASH 1 expression in a dose‐dependent manner. HEK ‐293T cells and NHEM s were transfected with different amounts of HA‐p53 plasmid as indicated. Exogenous SASH 1 RNA levels were measured by quantitative RT ‐ PCR and normalized to GAPDH . Expression of exogenous p53 protein and SASH 1 was analysed by Western blot along with GAPDH as a loading control. ( C ) and ( D ) Exogenous SASH 1 promotes expression of exogenous p53. Different amounts of GFP‐SASH1 plasmid and a certain amount of exogenous p53 were transfected to HEK ‐293T cells and NHEM s cells and plasmid. Increasing amounts of GFP ‐ SASH 1 trigger expression of exogenous p53, as analysed by QRT ‐ PCR and Western blot. ( E ) and ( F ) Exogenous SASH 1 promotes expression of endogenous p53. Different amounts of exogenous SASH 1 were introduced to HEK ‐293T cells and NHEM s. After 36‐hr transfection, cells were lysed and subjected to Western blot to analyse the expression of GFP ‐ SASH 1 as GAPDH as loading control. Results are the representative of three independent results. ( G ) An autoregulatory p53/ POMC /Gαs/ SASH 1 loop mediates reciprocal induction of p53 and SASH 1. p53 after being activated by different types of stress triggers the expression of POMC , Gαs and SASH 1 successively. The induced SASH 1 by p53/ POMC /Gαs cascade promotes the up‐regulation p53 in nucleus, then induced nucleic p53 conversely activates POMC /Gαs/ SASH 1 cascade, which consists an autoregulatory p53/ POMC /Gαs/ SASH 1 loop.
    Figure Legend Snippet: Reciprocal induction between p53 and SASH 1 is induced in normal cells. ( A ) and ( B ) Exogenous p53 ( HA ‐p53) triggers exogenous SASH 1 expression in a dose‐dependent manner. HEK ‐293T cells and NHEM s were transfected with different amounts of HA‐p53 plasmid as indicated. Exogenous SASH 1 RNA levels were measured by quantitative RT ‐ PCR and normalized to GAPDH . Expression of exogenous p53 protein and SASH 1 was analysed by Western blot along with GAPDH as a loading control. ( C ) and ( D ) Exogenous SASH 1 promotes expression of exogenous p53. Different amounts of GFP‐SASH1 plasmid and a certain amount of exogenous p53 were transfected to HEK ‐293T cells and NHEM s cells and plasmid. Increasing amounts of GFP ‐ SASH 1 trigger expression of exogenous p53, as analysed by QRT ‐ PCR and Western blot. ( E ) and ( F ) Exogenous SASH 1 promotes expression of endogenous p53. Different amounts of exogenous SASH 1 were introduced to HEK ‐293T cells and NHEM s. After 36‐hr transfection, cells were lysed and subjected to Western blot to analyse the expression of GFP ‐ SASH 1 as GAPDH as loading control. Results are the representative of three independent results. ( G ) An autoregulatory p53/ POMC /Gαs/ SASH 1 loop mediates reciprocal induction of p53 and SASH 1. p53 after being activated by different types of stress triggers the expression of POMC , Gαs and SASH 1 successively. The induced SASH 1 by p53/ POMC /Gαs cascade promotes the up‐regulation p53 in nucleus, then induced nucleic p53 conversely activates POMC /Gαs/ SASH 1 cascade, which consists an autoregulatory p53/ POMC /Gαs/ SASH 1 loop.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot

    8) Product Images from "Targeting posttranslational modifications of RIOK1 inhibits the progression of colorectal and gastric cancers"

    Article Title: Targeting posttranslational modifications of RIOK1 inhibits the progression of colorectal and gastric cancers

    Journal: eLife

    doi: 10.7554/eLife.29511

    T410 phosphorylation of RIOK1 antagonists SETD7-mediated K411 methylation, which stabilizes RIOK1 in CRC and GC cells. ( A ) The effect of knockdown of CK2α in HCT116 cells on the levels of RIOK1 proteins, T410p, and K411me. ( B ) The effect of the CK2α-K68A mutant on the levels of RIOK1 proteins, T410p, and K411me. ( C ) The effect of the CK2 kinase inhibitor TBB on the levels of RIOK1 proteins, T410p, and K411me. ( D ) The effect of CK2 knockdown in HCT116 cells on the RIOK1 protein stability. ( E ) CK2 inhibits K411me by SETD7 and its consequent RIOK1 ubiquitination in RKO cells. Indicated cells were cotransfected with HA-Ub and treated with MG132 for 12 hr. ( F ) Wild-type RIOK1 or the RIOK1-K411me1 mutant was overexpressed in cells, along with HA-ubiquitin and V5-SET7, in the presence or absence of the proteasome inhibitor MG132. Cell lysates were analyzed with indicated antibodies.
    Figure Legend Snippet: T410 phosphorylation of RIOK1 antagonists SETD7-mediated K411 methylation, which stabilizes RIOK1 in CRC and GC cells. ( A ) The effect of knockdown of CK2α in HCT116 cells on the levels of RIOK1 proteins, T410p, and K411me. ( B ) The effect of the CK2α-K68A mutant on the levels of RIOK1 proteins, T410p, and K411me. ( C ) The effect of the CK2 kinase inhibitor TBB on the levels of RIOK1 proteins, T410p, and K411me. ( D ) The effect of CK2 knockdown in HCT116 cells on the RIOK1 protein stability. ( E ) CK2 inhibits K411me by SETD7 and its consequent RIOK1 ubiquitination in RKO cells. Indicated cells were cotransfected with HA-Ub and treated with MG132 for 12 hr. ( F ) Wild-type RIOK1 or the RIOK1-K411me1 mutant was overexpressed in cells, along with HA-ubiquitin and V5-SET7, in the presence or absence of the proteasome inhibitor MG132. Cell lysates were analyzed with indicated antibodies.

    Techniques Used: Methylation, Mutagenesis

    CK2 phosphorylates RIOK1 at T410 both in vitro and in vivo. ( A ) RIOK1 consensus phosphorylation sites (shown in red) corresponding to the CK2 consensus motifs S/TXXE/D are presented. These consensus phosphorylation sites are exactly located within the consensus sequences of SETD7. ( B ) FLAG-RIOK1 was present in the HA-CK2 immunocomplex. Total cell lysates were extracted from 293T cells transiently co-transfected with FLAG-RIOK1 and HA-Vec and HA-CK2α subjected to immunoprecipitation with an anti-HA antibody followed by immunoblotting with indicated antibodies. ( C ) In vitro glutathione S -transferase (GST)-precipitation assay of no CK2α, or purified His-tagged CK2α combined with GST alone or GST-RIOK1. ( D ) Immunoprecipitation and immunoblot analysis of lysates of HEK293T cells expressing no plasmid (−) or plasmid encoding the Flag-tagged amino terminus (amino acids 1–242) of RIOK1 (Flag–RIOK1-N) or carboxyl terminus (amino acids 243–568) of RIOK1 (Flag–RIOK1-C), plus Myc-tagged CK2α, probed with anti-Myc and/or anti-Flag. ( E ) Lysates of HEK293T cells expressing Flag-tagged wild-type RIOK1 and Myc-tagged wild-type CK2α or CK2α-K68A; far right (λ-PPase), was analyzed using Western blot. GAPDH serves as a loading control throughout. ( F ) Immunoblot analysis of RIOK1 and CK2α in lysates of HEK293T cells expressing Flag-tagged wild-type RIOK1 or RIOK1 (T410A) and Myc-tagged wild-type CK2α or CK2α-K68A. ( G ) Immunoblot analysis of phosphorylated p-RIOK1-T410 and total RIOK1 and CK2 in lysates of HEK293T cells. ( H ) In vitro kinase assay of purified recombinant GST-tagged RIOK1 or RIOK1-T410A with HA-tagged CK2.
    Figure Legend Snippet: CK2 phosphorylates RIOK1 at T410 both in vitro and in vivo. ( A ) RIOK1 consensus phosphorylation sites (shown in red) corresponding to the CK2 consensus motifs S/TXXE/D are presented. These consensus phosphorylation sites are exactly located within the consensus sequences of SETD7. ( B ) FLAG-RIOK1 was present in the HA-CK2 immunocomplex. Total cell lysates were extracted from 293T cells transiently co-transfected with FLAG-RIOK1 and HA-Vec and HA-CK2α subjected to immunoprecipitation with an anti-HA antibody followed by immunoblotting with indicated antibodies. ( C ) In vitro glutathione S -transferase (GST)-precipitation assay of no CK2α, or purified His-tagged CK2α combined with GST alone or GST-RIOK1. ( D ) Immunoprecipitation and immunoblot analysis of lysates of HEK293T cells expressing no plasmid (−) or plasmid encoding the Flag-tagged amino terminus (amino acids 1–242) of RIOK1 (Flag–RIOK1-N) or carboxyl terminus (amino acids 243–568) of RIOK1 (Flag–RIOK1-C), plus Myc-tagged CK2α, probed with anti-Myc and/or anti-Flag. ( E ) Lysates of HEK293T cells expressing Flag-tagged wild-type RIOK1 and Myc-tagged wild-type CK2α or CK2α-K68A; far right (λ-PPase), was analyzed using Western blot. GAPDH serves as a loading control throughout. ( F ) Immunoblot analysis of RIOK1 and CK2α in lysates of HEK293T cells expressing Flag-tagged wild-type RIOK1 or RIOK1 (T410A) and Myc-tagged wild-type CK2α or CK2α-K68A. ( G ) Immunoblot analysis of phosphorylated p-RIOK1-T410 and total RIOK1 and CK2 in lysates of HEK293T cells. ( H ) In vitro kinase assay of purified recombinant GST-tagged RIOK1 or RIOK1-T410A with HA-tagged CK2.

    Techniques Used: In Vitro, In Vivo, Transfection, Immunoprecipitation, Purification, Expressing, Plasmid Preparation, Western Blot, Kinase Assay, Recombinant

    9) Product Images from "Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1"

    Article Title: Proliferating cell nuclear antigen interacts with the CRL4 ubiquitin ligase subunit CDT2 in DNA synthesis–induced degradation of CDT1

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.003049

    CDT2 contains a PIP box–like motif at its C terminus for PCNA interaction. A, the C-terminal region (amino acid residues 705–713) of CDT2 contains a conserved M XX I XX YF motif that resembles the PIP box motif in p21, CDT1, and DNA polymerase η. The conversion of Tyr-712 and Phe-713 to alanine (Ala) is shown. B, deletion or mutation of the C-terminal PIP box–like motif abolishes the interaction between CDT2 and PCNA. Deletion of the PIP (ΔPIP) or conversion of Tyr-712 and Phe-713 to alanine (YF/AA) in various N-terminal deletion mutants of CDT2 was expressed in bacteria, and their binding to PCNA was monitored. C , full-length GST–CDT2, CDT2 YF/AA mutant, and CUL4A were expressed in baculovirus-infected Sf9 cells and their interaction with PCNA was analyzed by GSH-Sepharose pulldown and blotting with anti-PCNA and GST antibodies.
    Figure Legend Snippet: CDT2 contains a PIP box–like motif at its C terminus for PCNA interaction. A, the C-terminal region (amino acid residues 705–713) of CDT2 contains a conserved M XX I XX YF motif that resembles the PIP box motif in p21, CDT1, and DNA polymerase η. The conversion of Tyr-712 and Phe-713 to alanine (Ala) is shown. B, deletion or mutation of the C-terminal PIP box–like motif abolishes the interaction between CDT2 and PCNA. Deletion of the PIP (ΔPIP) or conversion of Tyr-712 and Phe-713 to alanine (YF/AA) in various N-terminal deletion mutants of CDT2 was expressed in bacteria, and their binding to PCNA was monitored. C , full-length GST–CDT2, CDT2 YF/AA mutant, and CUL4A were expressed in baculovirus-infected Sf9 cells and their interaction with PCNA was analyzed by GSH-Sepharose pulldown and blotting with anti-PCNA and GST antibodies.

    Techniques Used: Mutagenesis, Binding Assay, Infection

    Recombinant CDT2 independently interacts with PCNA in vitro . A, CDK inhibitor p21 and CDT1 contain the PIP box that interacts with PCNA. The consensus PIP box: Q XX ψ XX θθ (ψ: hydrophobic amino acid residues, Leu, Val, Ile, Met; θ: aromatic amino acid residues, Phe and Tyr; X : any amino acid residues). B, GST–p21, GST–CDT2, GST–CDT1, and control GST proteins were purified from bacteria. The GST proteins were incubated with a bacterial lysate containing recombinant human PCNA protein for 4 h at 4 °C. The protein complexes were isolated by GSH-Sepharose and Western blotted with anti-PCNA and GST antibodies. C, GST–p21, GST–p27, GST–CUL4A, and GST–CDT2 were isolated from Sf9 cells infected with baculoviral constructs of GST proteins. The GST proteins (1 μg each) were incubated with bacterial lysates containing expressed recombinant human PCNA protein as in A and the proteins pulled down by the GSH-Sepharose were analyzed by anti-PCNA and anti-GST antibodies.
    Figure Legend Snippet: Recombinant CDT2 independently interacts with PCNA in vitro . A, CDK inhibitor p21 and CDT1 contain the PIP box that interacts with PCNA. The consensus PIP box: Q XX ψ XX θθ (ψ: hydrophobic amino acid residues, Leu, Val, Ile, Met; θ: aromatic amino acid residues, Phe and Tyr; X : any amino acid residues). B, GST–p21, GST–CDT2, GST–CDT1, and control GST proteins were purified from bacteria. The GST proteins were incubated with a bacterial lysate containing recombinant human PCNA protein for 4 h at 4 °C. The protein complexes were isolated by GSH-Sepharose and Western blotted with anti-PCNA and GST antibodies. C, GST–p21, GST–p27, GST–CUL4A, and GST–CDT2 were isolated from Sf9 cells infected with baculoviral constructs of GST proteins. The GST proteins (1 μg each) were incubated with bacterial lysates containing expressed recombinant human PCNA protein as in A and the proteins pulled down by the GSH-Sepharose were analyzed by anti-PCNA and anti-GST antibodies.

    Techniques Used: Recombinant, In Vitro, Purification, Incubation, Isolation, Western Blot, Infection, Construct

    Localization of the region in CDT2 for PCNA interaction. A, schematic illustration of the deletion mutants of the CDT2 protein and their ability to interact with PCNA. B and C , the C-terminal region (600–730) of CDT2 interacts with PCNA. GST-CDT2 and various N-terminal or C-terminal deletion constructs were expressed in bacteria and purified. They were incubated with bacterial lysates recombinant PCNA. The interactions were analyzed in GSH-Sepharose pulldown and blotting with anti-PCNA and GST antibodies.
    Figure Legend Snippet: Localization of the region in CDT2 for PCNA interaction. A, schematic illustration of the deletion mutants of the CDT2 protein and their ability to interact with PCNA. B and C , the C-terminal region (600–730) of CDT2 interacts with PCNA. GST-CDT2 and various N-terminal or C-terminal deletion constructs were expressed in bacteria and purified. They were incubated with bacterial lysates recombinant PCNA. The interactions were analyzed in GSH-Sepharose pulldown and blotting with anti-PCNA and GST antibodies.

    Techniques Used: Construct, Purification, Incubation, Recombinant

    A model for DNA replication-dependent degradation of CDT1 by trimeric PCNA and CDT2. A, both CDT2 and CDT1 interact with PCNA through their respective PIP box motifs. During DNA replication or DNA repair synthesis, the trimeric form of PCNA brings CDT1 and CDT2 together to the same PCNA clamp. B, the proximity of CDT1 and CDT2 leads to the polyubiquitination and eventual proteolysis of CDT1 by the CRL4 DCAT2 ubiquitin E3 ligase complex in response to DNA replication and DNA damage.
    Figure Legend Snippet: A model for DNA replication-dependent degradation of CDT1 by trimeric PCNA and CDT2. A, both CDT2 and CDT1 interact with PCNA through their respective PIP box motifs. During DNA replication or DNA repair synthesis, the trimeric form of PCNA brings CDT1 and CDT2 together to the same PCNA clamp. B, the proximity of CDT1 and CDT2 leads to the polyubiquitination and eventual proteolysis of CDT1 by the CRL4 DCAT2 ubiquitin E3 ligase complex in response to DNA replication and DNA damage.

    Techniques Used:

    10) Product Images from "Mixed lineage leukaemia histone methylases 1 collaborate with ERα to regulate HOXA10 expression in AML"

    Article Title: Mixed lineage leukaemia histone methylases 1 collaborate with ERα to regulate HOXA10 expression in AML

    Journal: Bioscience Reports

    doi: 10.1042/BSR20140116

    The interaction of MLL1 and ERs of HOXA10 promoter HL-60 and THP-1 cells were treated with 100 nM E2 for 8 h before being harvested for preparation of nuclear extract. The extracts were immunoprecipitated by using MLL1 antibody. The immunoprecipitated MLL1 complexes were then analysed by Western blot, using ERa antibodies. Immunoprecipitation with protein G agarose beads was used as negative control.
    Figure Legend Snippet: The interaction of MLL1 and ERs of HOXA10 promoter HL-60 and THP-1 cells were treated with 100 nM E2 for 8 h before being harvested for preparation of nuclear extract. The extracts were immunoprecipitated by using MLL1 antibody. The immunoprecipitated MLL1 complexes were then analysed by Western blot, using ERa antibodies. Immunoprecipitation with protein G agarose beads was used as negative control.

    Techniques Used: Immunoprecipitation, Western Blot, Negative Control

    The difference of H3K4 methylation status, HOXA10 promoter methylation status and HOXA10 expression with and without MLL1 knockdown HL-60 was grown up to 60% confluency and then separately transfected with phosphorothioate oligonucleotides specific for MLL1 phosphorothioate oligonucleotides using Lipofectamine 2000 (Invitrogen). Control cells were treated with a scramble antisense oligonucleotide with no homology with the MLL1, MLL2, MLL3 or MLL4 gene. The antisense oligonucleotide-transfected cells were incubated for 24 h and then cells were harvested and DNA, total RNA and protein was isolated and analysed by MSP, RT–PCR and Western-blot to test HOXA10 promoter methylation status with and without MLL1 knockdown ( A ), Lane L was 1000-bp DNA ladder. Lane M and U referred to methylation and unmethylation bands of MSP, respectively. The gel images showed the promoter CpG methylation of HOXA10 promoter in acute leukaemia cell line. ( B ) H3K4 methylation status with and without MLL1 knockdown. HOXA10 expression level with and without MLL1 knockdown in mRNA ( C ) and protein ( D ).
    Figure Legend Snippet: The difference of H3K4 methylation status, HOXA10 promoter methylation status and HOXA10 expression with and without MLL1 knockdown HL-60 was grown up to 60% confluency and then separately transfected with phosphorothioate oligonucleotides specific for MLL1 phosphorothioate oligonucleotides using Lipofectamine 2000 (Invitrogen). Control cells were treated with a scramble antisense oligonucleotide with no homology with the MLL1, MLL2, MLL3 or MLL4 gene. The antisense oligonucleotide-transfected cells were incubated for 24 h and then cells were harvested and DNA, total RNA and protein was isolated and analysed by MSP, RT–PCR and Western-blot to test HOXA10 promoter methylation status with and without MLL1 knockdown ( A ), Lane L was 1000-bp DNA ladder. Lane M and U referred to methylation and unmethylation bands of MSP, respectively. The gel images showed the promoter CpG methylation of HOXA10 promoter in acute leukaemia cell line. ( B ) H3K4 methylation status with and without MLL1 knockdown. HOXA10 expression level with and without MLL1 knockdown in mRNA ( C ) and protein ( D ).

    Techniques Used: Methylation, Expressing, Transfection, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, CpG Methylation Assay

    Effect of MLLs on expression of HOXA10 HL-60 and THP-1 cells were grown up to 60% confluency and then separately transfected with phosphorothioate oligonucleotides specific for MLL1, MLL2, MLL3 and MLL4 phosphorothioate oligonucleotides using Lipofectamine 2000 (Invitrogen). Control cells were treated with a scramble antisense oligonucleotide with no homology with the MLL1, MLL2, MLL3 or MLL4 gene. The antisense oligonucleotide-transfected cells were incubated for 24 h, and then cells were harvested and total RNA and protein was isolated and analysed by RT–PCR and Western blot. The t test was used to examine the difference between groups.* P
    Figure Legend Snippet: Effect of MLLs on expression of HOXA10 HL-60 and THP-1 cells were grown up to 60% confluency and then separately transfected with phosphorothioate oligonucleotides specific for MLL1, MLL2, MLL3 and MLL4 phosphorothioate oligonucleotides using Lipofectamine 2000 (Invitrogen). Control cells were treated with a scramble antisense oligonucleotide with no homology with the MLL1, MLL2, MLL3 or MLL4 gene. The antisense oligonucleotide-transfected cells were incubated for 24 h, and then cells were harvested and total RNA and protein was isolated and analysed by RT–PCR and Western blot. The t test was used to examine the difference between groups.* P

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

    E2-dependent recruitment of ERa and MLL1 in ERE1 and ERE2 of the HOXA10 promoter HL-60 and THP-1 were treated with 100 nM E2 for 8 h. Afterwards, the control and E2-treated cells were subjected to ChIP analysis using antibodies against MLL1 and ERα. β-Actin antibody was used as control IgG. The immunoprecipitated DNA fragments were PCR amplified using primers specific for ERE1 and ERE2 of the HOXA10 promoter.
    Figure Legend Snippet: E2-dependent recruitment of ERa and MLL1 in ERE1 and ERE2 of the HOXA10 promoter HL-60 and THP-1 were treated with 100 nM E2 for 8 h. Afterwards, the control and E2-treated cells were subjected to ChIP analysis using antibodies against MLL1 and ERα. β-Actin antibody was used as control IgG. The immunoprecipitated DNA fragments were PCR amplified using primers specific for ERE1 and ERE2 of the HOXA10 promoter.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Amplification

    11) Product Images from "Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer"

    Article Title: Histone methyltransferase SETD1A interacts with HIF1α to enhance glycolysis and promote cancer progression in gastric cancer

    Journal: Molecular Oncology

    doi: 10.1002/1878-0261.12689

    The expression of SETD1A is positively correlated with the expression of glycolytic genes in human gastric cancer specimens from TCGA dataset. Linear regression of SETD1A and glycolytic genes GLUT1 (A), HK2 (B), PFK2 (C), PKM (D), and MCT4 (E) using GC samples from TCGA database obtained by GEPIA.
    Figure Legend Snippet: The expression of SETD1A is positively correlated with the expression of glycolytic genes in human gastric cancer specimens from TCGA dataset. Linear regression of SETD1A and glycolytic genes GLUT1 (A), HK2 (B), PFK2 (C), PKM (D), and MCT4 (E) using GC samples from TCGA database obtained by GEPIA.

    Techniques Used: Expressing

    SETD1A promotes gastric cancer cell proliferation. (A, B) Overexpression of SETD1A increased BGC‐823 and AGS cell proliferation. BGC‐823 and AGS cells were transfected with SETD1A plasmid for 48 h and then seeded into 96‐well plates for CCK‐8 assays (mean ± SEM; n = 4; Student’s t ‐test). (C, D) Knockdown of SETD1A decreased BGC‐823 and AGS cell proliferation. Cells were transfected with SETD1A siRNAs for 48 h and then seeded into 96‐well plates for CCK‐8 assays (mean ± SEM; n = 4; Student’s t ‐test). (E, F) Stable SETD1A‐knockdown BGC‐823 and AGS cells grown slowly compared to control cells (mean ± SEM; n = 4; Student’s t ‐test). (mean ± SEM; n = 4; Student’s t ‐test) * P
    Figure Legend Snippet: SETD1A promotes gastric cancer cell proliferation. (A, B) Overexpression of SETD1A increased BGC‐823 and AGS cell proliferation. BGC‐823 and AGS cells were transfected with SETD1A plasmid for 48 h and then seeded into 96‐well plates for CCK‐8 assays (mean ± SEM; n = 4; Student’s t ‐test). (C, D) Knockdown of SETD1A decreased BGC‐823 and AGS cell proliferation. Cells were transfected with SETD1A siRNAs for 48 h and then seeded into 96‐well plates for CCK‐8 assays (mean ± SEM; n = 4; Student’s t ‐test). (E, F) Stable SETD1A‐knockdown BGC‐823 and AGS cells grown slowly compared to control cells (mean ± SEM; n = 4; Student’s t ‐test). (mean ± SEM; n = 4; Student’s t ‐test) * P

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, CCK-8 Assay

    SETD1A cooperates with HIF1α to enhance HIF1α transactivation. (A) SETD1A cooperated with HIF1α to enhance the HK2 and PFK2 promoter–reporter activity (mean ± SEM; n = 3; Student’s t ‐test). (B) Downregulation of SETD1A decreased the HK2 and PFK2 promoter activity in BGC‐823 cells. SETD1A‐knockdown and control BGC‐823 cells were transfected with HRE‐Luc under normoxic (N) and hypoxic (H) conditions. Luciferase activity was measured 48 h after transfection (mean ± SEM; n = 3; Student’s t ‐test). (C) Co‐IP analysis of the interaction between Flag‐SETD1A and HA‐HIF1α. (D) Co‐IP analysis of the interaction between endogenous SETD1A and HIF1α in BGC‐823 cells. (E) Downregulation of SETD1A did not change the expression of HIF1α and global H3K4me3 protein in BGC‐823 cells. (F, G) Downregulation of SETD1A reduced the levels of HIF1α (F), SETD1A (G), and H3K4me3 (H) on the HK2 and PFK2 promoter in BGC‐823 cells. ChIP analysis of the levels of HIF1α (F), SETD1A (G), and H3K4me3 (H) on the HK2 and PFK2 promoter in SETD1A‐knockdown and control BGC‐823 cells under normoxic (N) or hypoxic (H) condition (mean ± SEM; n = 3; Student’s t ‐test). * P
    Figure Legend Snippet: SETD1A cooperates with HIF1α to enhance HIF1α transactivation. (A) SETD1A cooperated with HIF1α to enhance the HK2 and PFK2 promoter–reporter activity (mean ± SEM; n = 3; Student’s t ‐test). (B) Downregulation of SETD1A decreased the HK2 and PFK2 promoter activity in BGC‐823 cells. SETD1A‐knockdown and control BGC‐823 cells were transfected with HRE‐Luc under normoxic (N) and hypoxic (H) conditions. Luciferase activity was measured 48 h after transfection (mean ± SEM; n = 3; Student’s t ‐test). (C) Co‐IP analysis of the interaction between Flag‐SETD1A and HA‐HIF1α. (D) Co‐IP analysis of the interaction between endogenous SETD1A and HIF1α in BGC‐823 cells. (E) Downregulation of SETD1A did not change the expression of HIF1α and global H3K4me3 protein in BGC‐823 cells. (F, G) Downregulation of SETD1A reduced the levels of HIF1α (F), SETD1A (G), and H3K4me3 (H) on the HK2 and PFK2 promoter in BGC‐823 cells. ChIP analysis of the levels of HIF1α (F), SETD1A (G), and H3K4me3 (H) on the HK2 and PFK2 promoter in SETD1A‐knockdown and control BGC‐823 cells under normoxic (N) or hypoxic (H) condition (mean ± SEM; n = 3; Student’s t ‐test). * P

    Techniques Used: Activity Assay, Transfection, Luciferase, Co-Immunoprecipitation Assay, Expressing, Chromatin Immunoprecipitation

    SETD1A was overexpressed in human GC specimens and predicated poor outcome. (A) Analysis of the protein of SETD1A in 18 pairs of GC specimens and surrounding nontumor tissues. (B) Analysis of the expression of SETD1A in normal ( n = 36) and GC ( n = 408) specimens from TCGA dataset obtained by GEPIA. (C) Analysis of the overall survival rate in SETD1A‐high and SETD1A‐low expression GC patients from GEO dataset. * P
    Figure Legend Snippet: SETD1A was overexpressed in human GC specimens and predicated poor outcome. (A) Analysis of the protein of SETD1A in 18 pairs of GC specimens and surrounding nontumor tissues. (B) Analysis of the expression of SETD1A in normal ( n = 36) and GC ( n = 408) specimens from TCGA dataset obtained by GEPIA. (C) Analysis of the overall survival rate in SETD1A‐high and SETD1A‐low expression GC patients from GEO dataset. * P

    Techniques Used: Expressing

    Downregulation of SETD1A decreases glycolysis in BGC‐823 cells. (A–C) Glucose uptake (A), lactate production (B), and pH values (C) of SETD1A‐knockdown cells were reduced under normoxic (N) and hypoxic (H) conditions for 24 h compared to control BGC‐823 cells (mean ± SEM; n = 3; Student’s t ‐test). (D) The protein levels of HK2 and LDHA were remarkedly reduced in SETD1A‐knockdown BGC‐823 cells under normoxic (N) and hypoxic (H) conditions for 24 h. (E–J) The mRNA levels of GLUT1 (E), HK2 (F), PFK2 (G), PKM2 (H), LDHA (I), and MCT4 (J) were remarkedly reduced in SETD1A‐knockdown BGC‐823 cells under normoxic (N) and hypoxic (H) conditions for 24 h (mean ± SEM; n = 4; Student’s t ‐test). * P
    Figure Legend Snippet: Downregulation of SETD1A decreases glycolysis in BGC‐823 cells. (A–C) Glucose uptake (A), lactate production (B), and pH values (C) of SETD1A‐knockdown cells were reduced under normoxic (N) and hypoxic (H) conditions for 24 h compared to control BGC‐823 cells (mean ± SEM; n = 3; Student’s t ‐test). (D) The protein levels of HK2 and LDHA were remarkedly reduced in SETD1A‐knockdown BGC‐823 cells under normoxic (N) and hypoxic (H) conditions for 24 h. (E–J) The mRNA levels of GLUT1 (E), HK2 (F), PFK2 (G), PKM2 (H), LDHA (I), and MCT4 (J) were remarkedly reduced in SETD1A‐knockdown BGC‐823 cells under normoxic (N) and hypoxic (H) conditions for 24 h (mean ± SEM; n = 4; Student’s t ‐test). * P

    Techniques Used:

    Knockdown of SETD1A reduces gastric cancer cell tumorigenesis. (A, B) Downregulation of SETD1A in BGC‐823 cells inhibited xenograft tumor growth (A) in nude mice and reduced tumor weight (B) (mean ± SEM; n = 6; Student’s t ‐test). (C) The mRNA levels of the GLUT1, HK2, PFK2, PKM2, LDHA, and MCT4 were reduced in SETD1A‐knockdown tumors (mean ± SEM; n = 6; Student’s t ‐test). * P
    Figure Legend Snippet: Knockdown of SETD1A reduces gastric cancer cell tumorigenesis. (A, B) Downregulation of SETD1A in BGC‐823 cells inhibited xenograft tumor growth (A) in nude mice and reduced tumor weight (B) (mean ± SEM; n = 6; Student’s t ‐test). (C) The mRNA levels of the GLUT1, HK2, PFK2, PKM2, LDHA, and MCT4 were reduced in SETD1A‐knockdown tumors (mean ± SEM; n = 6; Student’s t ‐test). * P

    Techniques Used: Mouse Assay

    Inhibition of HIF1α suppresses SETD1A‐enhanced GC cell proliferation. (A, B) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced BGC‐823 cell proliferation (mean ± SEM; n = 3; Student’s t ‐test). (C, D) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced AGS cell proliferation (mean ± SEM; n = 4; Student’s t ‐test). ** P
    Figure Legend Snippet: Inhibition of HIF1α suppresses SETD1A‐enhanced GC cell proliferation. (A, B) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced BGC‐823 cell proliferation (mean ± SEM; n = 3; Student’s t ‐test). (C, D) Knockdown of HIF1α suppressed glycolytic pathway and SETD1A‐enhanced AGS cell proliferation (mean ± SEM; n = 4; Student’s t ‐test). ** P

    Techniques Used: Inhibition

    12) Product Images from "Single Agent and Synergistic Activity of the "First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma"

    Article Title: Single Agent and Synergistic Activity of the "First-in-Class" Dual PI3K/BRD4 Inhibitor SF1126 with Sorafenib in Hepatocellular Carcinoma

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-15-0976

    SF1126 displaces BRD4 from the MYC transcriptional start site in HCC. A,
    Figure Legend Snippet: SF1126 displaces BRD4 from the MYC transcriptional start site in HCC. A,

    Techniques Used:

    13) Product Images from "Dual-activity PI3K–BRD4 inhibitor for the orthogonal inhibition of MYC to block tumor growth and metastasis"

    Article Title: Dual-activity PI3K–BRD4 inhibitor for the orthogonal inhibition of MYC to block tumor growth and metastasis

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

    doi: 10.1073/pnas.1613091114

    IC 50 of indicated PI3K inhibitors for H4K5acK8acK12acK16ac-bound BD1 and BD2 of BRD4.
    Figure Legend Snippet: IC 50 of indicated PI3K inhibitors for H4K5acK8acK12acK16ac-bound BD1 and BD2 of BRD4.

    Techniques Used:

    Morpholinothienopyrane is an inhibitor of BDs. ( A and B ) IC 50 values, measured by displacement binding assays, show that SF2523 and its derivatives are inhibitors of BRD4 BDs. IC 50 values, measured by a kinase screening assay, are shown in the second
    Figure Legend Snippet: Morpholinothienopyrane is an inhibitor of BDs. ( A and B ) IC 50 values, measured by displacement binding assays, show that SF2523 and its derivatives are inhibitors of BRD4 BDs. IC 50 values, measured by a kinase screening assay, are shown in the second

    Techniques Used: Binding Assay, Screening Assay

    PI3K-specific inhibitor targets BRD4. ( A ) RT-PCR data showing the effect of indicated inhibitors on MYCN expression in neuroblastoma SKNBE2 cells. SKNBE2 cells were serum-starved for 4 h, stimulated with 50 ng/mL IGF, and treated with 1 µM JQ1,
    Figure Legend Snippet: PI3K-specific inhibitor targets BRD4. ( A ) RT-PCR data showing the effect of indicated inhibitors on MYCN expression in neuroblastoma SKNBE2 cells. SKNBE2 cells were serum-starved for 4 h, stimulated with 50 ng/mL IGF, and treated with 1 µM JQ1,

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Structural mechanism for the recognition of inhibitors by BRD4 BD2. ( A ) The crystal structure of BRD4 BD2 (yellow) in complex with SF2558HA (pink). ( B ) Structural overlay of the complexes: BRD4 BD2 (yellow) with SF2558HA (pink), BRD4 BD1 (green) with
    Figure Legend Snippet: Structural mechanism for the recognition of inhibitors by BRD4 BD2. ( A ) The crystal structure of BRD4 BD2 (yellow) in complex with SF2558HA (pink). ( B ) Structural overlay of the complexes: BRD4 BD2 (yellow) with SF2558HA (pink), BRD4 BD1 (green) with

    Techniques Used:

    Superimposed 1 H, 15 N HSQC spectra of BRD4 BD2 in the apo-state (black) and bound to SF2558HA (blue), SF2523 (green), and SF2535 (red).
    Figure Legend Snippet: Superimposed 1 H, 15 N HSQC spectra of BRD4 BD2 in the apo-state (black) and bound to SF2558HA (blue), SF2523 (green), and SF2535 (red).

    Techniques Used:

    Structural Mechanism for BRD4 BD1 Inhibition by TP Compounds.
    Figure Legend Snippet: Structural Mechanism for BRD4 BD1 Inhibition by TP Compounds.

    Techniques Used: Inhibition

    Structural mechanism for the recognition of inhibitors by BRD4 BD1. ( A and B ) The crystal structure of BRD4 BD1 (green) in complex with SF2523 (yellow). Water molecules and hydrogen bonds are shown as yellow dashes and red spheres, respectively. ( C ) Overlay
    Figure Legend Snippet: Structural mechanism for the recognition of inhibitors by BRD4 BD1. ( A and B ) The crystal structure of BRD4 BD1 (green) in complex with SF2523 (yellow). Water molecules and hydrogen bonds are shown as yellow dashes and red spheres, respectively. ( C ) Overlay

    Techniques Used:

    14) Product Images from "TRPM2 channel–mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK-signaling pathway"

    Article Title: TRPM2 channel–mediated regulation of autophagy maintains mitochondrial function and promotes gastric cancer cell survival via the JNK-signaling pathway

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.817635

    Expression level of TRPM2 is negatively correlated with the overall survival rate of gastric cancer patients. The expression of TRPM2 was analyzed according to the Kaplan-Meier method using a median cutoff. Patients with TRPM2 mRNA levels higher than the median value were considered “high,” and patients with mRNA expression lower than the median were classified as “low.” Survival curves show the correlation between high TRPM2 expression and low patient survival. A, all patients; B, patients with stage I and II cancer and patients with stage III and IV gastric cancer. The hazard ratios generated are greater than 1 suggesting that patients with high TRPM2 expression will die at a higher rate in a given period of time than those with low TRPM2.
    Figure Legend Snippet: Expression level of TRPM2 is negatively correlated with the overall survival rate of gastric cancer patients. The expression of TRPM2 was analyzed according to the Kaplan-Meier method using a median cutoff. Patients with TRPM2 mRNA levels higher than the median value were considered “high,” and patients with mRNA expression lower than the median were classified as “low.” Survival curves show the correlation between high TRPM2 expression and low patient survival. A, all patients; B, patients with stage I and II cancer and patients with stage III and IV gastric cancer. The hazard ratios generated are greater than 1 suggesting that patients with high TRPM2 expression will die at a higher rate in a given period of time than those with low TRPM2.

    Techniques Used: Expressing, Generated

    Down-regulation of TRPM2 promotes cell death in AGS and MKN-45 cells. A , annexin V/7AAD staining of TRPM2 KD and Scr. cells 72 h after being seeded in 6-well plates. Dot plots represent the population of live cells ( lower left quadrant ), necrotic cells ( upper left quadrant ), apoptotic cells ( lower right quadrant ), and early necrotic or late apoptotic cells ( upper right quadrant ). B, bar graphs depict the quantification of apoptosis data ( n = 3). C and D, Western blot analysis of cleaved caspase-7 in TRPM2 KD cells. Statistical significance of Western blotting results was calculated as the relative ratio of cleaved caspase-7 protein normalized to β-actin ( n = 3). Asterisks indicated a significant difference from scrambler: ***, p
    Figure Legend Snippet: Down-regulation of TRPM2 promotes cell death in AGS and MKN-45 cells. A , annexin V/7AAD staining of TRPM2 KD and Scr. cells 72 h after being seeded in 6-well plates. Dot plots represent the population of live cells ( lower left quadrant ), necrotic cells ( upper left quadrant ), apoptotic cells ( lower right quadrant ), and early necrotic or late apoptotic cells ( upper right quadrant ). B, bar graphs depict the quantification of apoptosis data ( n = 3). C and D, Western blot analysis of cleaved caspase-7 in TRPM2 KD cells. Statistical significance of Western blotting results was calculated as the relative ratio of cleaved caspase-7 protein normalized to β-actin ( n = 3). Asterisks indicated a significant difference from scrambler: ***, p

    Techniques Used: Staining, Western Blot

    TRPM2 KD inhibits proliferation in AGS and MKN-45 cells. A and C, trypan blue counting of Scr. and TRPM2 knockdown cells at 24, 48, and 72 h after being seeded ( n = 4). B and D, MTT assay was used to quantify viable cells at 24, 48, and 72 h after being seeded ( n = 5). E and G, CFSE proliferation assay after 4 days of cell culturing. In the corresponding histograms, the x axis represents the CFSE fluorescent signal intensity, and the y axis shows the number of events. F and H, bar graphs representing CFSE mean of data from n = 3; ***, p
    Figure Legend Snippet: TRPM2 KD inhibits proliferation in AGS and MKN-45 cells. A and C, trypan blue counting of Scr. and TRPM2 knockdown cells at 24, 48, and 72 h after being seeded ( n = 4). B and D, MTT assay was used to quantify viable cells at 24, 48, and 72 h after being seeded ( n = 5). E and G, CFSE proliferation assay after 4 days of cell culturing. In the corresponding histograms, the x axis represents the CFSE fluorescent signal intensity, and the y axis shows the number of events. F and H, bar graphs representing CFSE mean of data from n = 3; ***, p

    Techniques Used: MTT Assay, Proliferation Assay, Cell Culture

    15) Product Images from "Abnormal Levels of Gadd45alpha in Developing Neocortex Impair Neurite Outgrowth"

    Article Title: Abnormal Levels of Gadd45alpha in Developing Neocortex Impair Neurite Outgrowth

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044207

    Gadd45a expression is critical for development of normal neuron morphology in neocortex. E15.5 mouse cerebral cortex was electroporated with constructs designed to reduce or increase Gadd45a levels in electroporated cells. Each construct was co-electroporated with vectors encoding red (RFP) or green (GFP) fluorescent reporter protein. RFP or GFP-positive neurons were analyzed at P14 or P10 respectively (n = 4–6 brains/group from two separate litters). ( A and B ) Although most Gadd45a shRNA-treated neurons ( B ) reached the upper layers of neocortex, there were significantly more cells distributed beneath upper layers compared to RFP alone ( A ) (also see Fig. S8 ). Higher magnification of control neurons ( A’ and A” ) and Gadd45a shRNA-treated ( B’ and B” ) reveals that the dendritic processes of neurons expressing Gadd45a shRNA are less arborized than those of neurons expressing RFP alone. Arrows indicate somas of RFP positive neurons. ( C ) Migration of neurons to the upper layers (2/3) of neocortex is unaffected by overexpression of Gadd45a. Left panels show neurons in control brains. Right panels show neurons overexpressing Gadd45a. ( C’ ) Higher magnification images show examples of Gadd45a-AU1 neurons (right panels) with hypertrophied, multipolar shaped somas compared to control neurons (left panels). Scale bars = 50 µm ( A, A” ).
    Figure Legend Snippet: Gadd45a expression is critical for development of normal neuron morphology in neocortex. E15.5 mouse cerebral cortex was electroporated with constructs designed to reduce or increase Gadd45a levels in electroporated cells. Each construct was co-electroporated with vectors encoding red (RFP) or green (GFP) fluorescent reporter protein. RFP or GFP-positive neurons were analyzed at P14 or P10 respectively (n = 4–6 brains/group from two separate litters). ( A and B ) Although most Gadd45a shRNA-treated neurons ( B ) reached the upper layers of neocortex, there were significantly more cells distributed beneath upper layers compared to RFP alone ( A ) (also see Fig. S8 ). Higher magnification of control neurons ( A’ and A” ) and Gadd45a shRNA-treated ( B’ and B” ) reveals that the dendritic processes of neurons expressing Gadd45a shRNA are less arborized than those of neurons expressing RFP alone. Arrows indicate somas of RFP positive neurons. ( C ) Migration of neurons to the upper layers (2/3) of neocortex is unaffected by overexpression of Gadd45a. Left panels show neurons in control brains. Right panels show neurons overexpressing Gadd45a. ( C’ ) Higher magnification images show examples of Gadd45a-AU1 neurons (right panels) with hypertrophied, multipolar shaped somas compared to control neurons (left panels). Scale bars = 50 µm ( A, A” ).

    Techniques Used: Expressing, Construct, shRNA, Migration, Over Expression

    Gadd45a regulates neurite outgrowth from cortical neurons in vitro. ( A ) Method used to transfect, culture and analyze transfected cortical neurons. E15.5 mice were electroporated with cDNA encoding GFP and different Gadd45a constructs. At E16.5, GFP-positive regions of cortex were isolated, cultured 6DIV, fixed, imaged and analyzed. Sholl analyses were performed by centering concentric rings with increasing radii of 10 um over the soma center and counting the number of times the dendritic processes intersected the rings. ( B-D ) Images of GFP-positive cells converted for Sholl analyses. Examples of control (pLKO +GFP) ( B ), Gadd45a knockdown (Gadd45a shRNA + GFP) ( C ), and Gadd45a overexpressing (Gadd45a -AU1+GFP) ( D ) neurons. Scale bar = 20 µm. ( C and E ) Gadd45a knockdown results in significantly fewer neurites. ( D and F ) Gadd45a overexpression results in hypertrophied somas, and significantly fewer distal processes (∼70 µm –200 um from the soma). The significant difference within the first 10 µm is likely due to the increase in soma size (see Fig. S6 ). Note: Gadd45a overexpression (blue) was compared to two different controls, GFP (green) and pLKO (black, same data presented in (E)). ( E ) Electroporation of a Gadd45a-shRNA resistant construct (R- Gadd45a (blue line)) is able to significantly restore neuronal morphology compared to Gadd45a shRNA treated neurons. ( E and F ) Lines with asterisks indicate ranges where data was averaged for posthoc analysis (bar graphs). Sholl data was analyzed using two-way ANOVA with repeated measures while the averaged data was analyzed by Fisher’s PLSD post-hoc test. ns = not significant.
    Figure Legend Snippet: Gadd45a regulates neurite outgrowth from cortical neurons in vitro. ( A ) Method used to transfect, culture and analyze transfected cortical neurons. E15.5 mice were electroporated with cDNA encoding GFP and different Gadd45a constructs. At E16.5, GFP-positive regions of cortex were isolated, cultured 6DIV, fixed, imaged and analyzed. Sholl analyses were performed by centering concentric rings with increasing radii of 10 um over the soma center and counting the number of times the dendritic processes intersected the rings. ( B-D ) Images of GFP-positive cells converted for Sholl analyses. Examples of control (pLKO +GFP) ( B ), Gadd45a knockdown (Gadd45a shRNA + GFP) ( C ), and Gadd45a overexpressing (Gadd45a -AU1+GFP) ( D ) neurons. Scale bar = 20 µm. ( C and E ) Gadd45a knockdown results in significantly fewer neurites. ( D and F ) Gadd45a overexpression results in hypertrophied somas, and significantly fewer distal processes (∼70 µm –200 um from the soma). The significant difference within the first 10 µm is likely due to the increase in soma size (see Fig. S6 ). Note: Gadd45a overexpression (blue) was compared to two different controls, GFP (green) and pLKO (black, same data presented in (E)). ( E ) Electroporation of a Gadd45a-shRNA resistant construct (R- Gadd45a (blue line)) is able to significantly restore neuronal morphology compared to Gadd45a shRNA treated neurons. ( E and F ) Lines with asterisks indicate ranges where data was averaged for posthoc analysis (bar graphs). Sholl data was analyzed using two-way ANOVA with repeated measures while the averaged data was analyzed by Fisher’s PLSD post-hoc test. ns = not significant.

    Techniques Used: In Vitro, Transfection, Mouse Assay, Construct, Isolation, Cell Culture, shRNA, Over Expression, Electroporation

    16) Product Images from "Antibody crossreactivity between the tumour suppressor PHLPP1 and the proto-oncogene ?-catenin"

    Article Title: Antibody crossreactivity between the tumour suppressor PHLPP1 and the proto-oncogene ?-catenin

    Journal: EMBO Reports

    doi: 10.1038/embor.2012.188

    PH domain leucine-rich repeat protein phosphatase 1 antibodies crossreact with β-catenin. ( A ) Characterization of immunoreactivity of the PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) antibody Bethyl A300-660A indicates the presence
    Figure Legend Snippet: PH domain leucine-rich repeat protein phosphatase 1 antibodies crossreact with β-catenin. ( A ) Characterization of immunoreactivity of the PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) antibody Bethyl A300-660A indicates the presence

    Techniques Used:

    17) Product Images from "Combination of triapine, olaparib, and cediranib suppresses progression of BRCA-wild type and PARP inhibitor-resistant epithelial ovarian cancer"

    Article Title: Combination of triapine, olaparib, and cediranib suppresses progression of BRCA-wild type and PARP inhibitor-resistant epithelial ovarian cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207399

    Characterization of intraperitoneal EOC cell lines and xenografts. (A) Expression of BRCA2 in PEO1/4 cells and PEO1/4ip cells. Total protein was isolated from cells and subjected to western blot analysis for BRCA2 protein. HSC70 protein was used as a loading control. BRCA2 wild type and mutant bands are shown. (B, C) Sensitivity of PEO1/4 cells and PEO1/4ip cells to olaparib and paclitaxel. Cells were treated with various concentrations of olaparib or paclitaxel for 72 hr. MTS cytotoxicity assay was performed to determine percent survival relative to vehicle-treated controls. Data are means ± SE. (D, E) SCID-Beige mice were inoculated i.p. with PEO1ip or PEO4ip cells. After 3 days, mice were randomly assigned to 2 groups (n = 4) and treated i.p. with vehicle and olaparib (50 mg/kg) daily for 6 weeks (day 3 to 45). The body condition score (BCS) of mice bearing PEO1ip xenografts was monitored and the abdominal circumference of mice bearing PEO4ip xenografts was measured every 2–3 days to determine the endpoint and the Kaplan-Meier survival curve. p values were determined by the Mantel-Cox test compared with the control.
    Figure Legend Snippet: Characterization of intraperitoneal EOC cell lines and xenografts. (A) Expression of BRCA2 in PEO1/4 cells and PEO1/4ip cells. Total protein was isolated from cells and subjected to western blot analysis for BRCA2 protein. HSC70 protein was used as a loading control. BRCA2 wild type and mutant bands are shown. (B, C) Sensitivity of PEO1/4 cells and PEO1/4ip cells to olaparib and paclitaxel. Cells were treated with various concentrations of olaparib or paclitaxel for 72 hr. MTS cytotoxicity assay was performed to determine percent survival relative to vehicle-treated controls. Data are means ± SE. (D, E) SCID-Beige mice were inoculated i.p. with PEO1ip or PEO4ip cells. After 3 days, mice were randomly assigned to 2 groups (n = 4) and treated i.p. with vehicle and olaparib (50 mg/kg) daily for 6 weeks (day 3 to 45). The body condition score (BCS) of mice bearing PEO1ip xenografts was monitored and the abdominal circumference of mice bearing PEO4ip xenografts was measured every 2–3 days to determine the endpoint and the Kaplan-Meier survival curve. p values were determined by the Mantel-Cox test compared with the control.

    Techniques Used: Expressing, Isolation, Western Blot, Mutagenesis, Cytotoxicity Assay, Mouse Assay

    The effects of cediranib on AKT signaling and the sensitivity of BRCA2-wild type and mutated EOC cells to olaparib and triapine in vitro. PEO1 and PEO4 cells were treated with 1.25 μM cediranib, 0.75 μM triapine, or both drugs for 1 hr and then treated with various concentrations of olaparib for 72 hr. (A) MTS cytotoxicity assay was performed to determine percent survival relative to vehicle-treated controls. Data are means ± SD. (B) EOB was calculated to determine the effects of the combinations of cediranib, triapine, and olaparib on cell survival at all data points. EOB
    Figure Legend Snippet: The effects of cediranib on AKT signaling and the sensitivity of BRCA2-wild type and mutated EOC cells to olaparib and triapine in vitro. PEO1 and PEO4 cells were treated with 1.25 μM cediranib, 0.75 μM triapine, or both drugs for 1 hr and then treated with various concentrations of olaparib for 72 hr. (A) MTS cytotoxicity assay was performed to determine percent survival relative to vehicle-treated controls. Data are means ± SD. (B) EOB was calculated to determine the effects of the combinations of cediranib, triapine, and olaparib on cell survival at all data points. EOB

    Techniques Used: In Vitro, Cytotoxicity Assay

    18) Product Images from "Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage *"

    Article Title: Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.603423

    Centrobin overexpression results in increased cellular CPAP but not CP110 and hSAS-6 levels. A , 293T cells transfected with control or myc-centrobin expression vectors were lysed after 72 h of transfection and immunoblotted ( IB ) using the anti-CPAP, -CP110,
    Figure Legend Snippet: Centrobin overexpression results in increased cellular CPAP but not CP110 and hSAS-6 levels. A , 293T cells transfected with control or myc-centrobin expression vectors were lysed after 72 h of transfection and immunoblotted ( IB ) using the anti-CPAP, -CP110,

    Techniques Used: Over Expression, Transfection, Expressing

    19) Product Images from "Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity"

    Article Title: Reactivity of human AGO2 monoclonal antibody 11A9 with the SWI/SNF complex: A case study for rigorously defining antibody selectivity

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07539-4

    The 11A9 antibody may cross-react with SWI/SNF component SMARCC1/BAF155. ( A ) Stoichiometry analysis in HEK293T with iBAQ values generated with Maxquant normalized with the IgG isotype control. AGO2 is the bait and set at 1 as we used anti-AGO2 (11A9) for the IP. Error bars represent standard deviations of 3 individual IPs. ( B ) Label-free quantification of BAF155 A301-019A associated proteins compared to IgG control. Experiment was performed in triplicate in HEK293T cells. For the analysis a two sample T-test was applied. ( C ) Table with average Maxquant derived label free quantification intensities of 3 IP-MS experiments for BAF155 A301-019A and IgG with HEK293T wild type lysates. Mean LFQ values are times 10 billion and signals from AGO2 knock-out come from imputation. CV represents coefficient of variation. ( D ) Venn diagram representing the number of quantified and significant proteins in the SMARCC1 IP-MS and the AGO2 11A9 IP-MS and proteins that were quantified and significant in both experiments. ( E ) Stoichiometry analysis as in A with SMARCC1 as bait. SMARCC1 was set at 1 as we used anti-BAF155 (A301-019A) for the IP. Error bars represent standard deviations of 3 individual IPs. ( F ) Reciprocal co-IPs with AGO2 11A9 and BAF155 antibodies with anti-IgG as negative control in Wildtype and AGO2 knock-out HEK293T cells. All samples were loaded on the same gel and transferred to the same blot on which both the staining’s were performed with different detection methods. Uncropped images are shown in the supplementary.
    Figure Legend Snippet: The 11A9 antibody may cross-react with SWI/SNF component SMARCC1/BAF155. ( A ) Stoichiometry analysis in HEK293T with iBAQ values generated with Maxquant normalized with the IgG isotype control. AGO2 is the bait and set at 1 as we used anti-AGO2 (11A9) for the IP. Error bars represent standard deviations of 3 individual IPs. ( B ) Label-free quantification of BAF155 A301-019A associated proteins compared to IgG control. Experiment was performed in triplicate in HEK293T cells. For the analysis a two sample T-test was applied. ( C ) Table with average Maxquant derived label free quantification intensities of 3 IP-MS experiments for BAF155 A301-019A and IgG with HEK293T wild type lysates. Mean LFQ values are times 10 billion and signals from AGO2 knock-out come from imputation. CV represents coefficient of variation. ( D ) Venn diagram representing the number of quantified and significant proteins in the SMARCC1 IP-MS and the AGO2 11A9 IP-MS and proteins that were quantified and significant in both experiments. ( E ) Stoichiometry analysis as in A with SMARCC1 as bait. SMARCC1 was set at 1 as we used anti-BAF155 (A301-019A) for the IP. Error bars represent standard deviations of 3 individual IPs. ( F ) Reciprocal co-IPs with AGO2 11A9 and BAF155 antibodies with anti-IgG as negative control in Wildtype and AGO2 knock-out HEK293T cells. All samples were loaded on the same gel and transferred to the same blot on which both the staining’s were performed with different detection methods. Uncropped images are shown in the supplementary.

    Techniques Used: Generated, Derivative Assay, Mass Spectrometry, Knock-Out, Negative Control, Staining

    20) Product Images from "The Groucho-associated Phosphatase PPM1B Displaces Pax Transactivation Domain Interacting Protein (PTIP) to Switch the Transcription Factor Pax2 from a Transcriptional Activator to a Repressor *"

    Article Title: The Groucho-associated Phosphatase PPM1B Displaces Pax Transactivation Domain Interacting Protein (PTIP) to Switch the Transcription Factor Pax2 from a Transcriptional Activator to a Repressor *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.607424

    Grg4 and PPM1B co-IP. Rabbit anti-PPM1B or goat anti-Grg4 were used to IP proteins from nuclear extracts directly in the presence of EtBr and Western blotted ( WB ) as indicated. Controls are rabbit IgG for PPM1B and goat IgG for Grg4.
    Figure Legend Snippet: Grg4 and PPM1B co-IP. Rabbit anti-PPM1B or goat anti-Grg4 were used to IP proteins from nuclear extracts directly in the presence of EtBr and Western blotted ( WB ) as indicated. Controls are rabbit IgG for PPM1B and goat IgG for Grg4.

    Techniques Used: Co-Immunoprecipitation Assay, Western Blot

    PPM1B is required for Pax2- and Grg4-mediated gene repression. A , control negative sh GFP reporter cells or GFP reporter cells with shRNA 1, 2, or 4 were transfected with Grg4, Pax2, wild-type, or phosphatase-deficient PPM1B as indicated. Whole cell lysates were Western blotted ( WB ) for the indicated proteins with β-actin used as a loading control. Note that GFP expression increased upon Pax2 transfection ( lanes 2 , 7 , 12 , and 17 ) in all cell lines; this activation was inhibited by Grg4 ( lane 3 ) in control cells but not in PPM1B knockdown cells ( lanes 8 , 13 , and 18 ). The Grg4-mediated inhibition was rescued with wild-type PPM1B in the knockdown cells ( lanes 9 , 14 , and 19 ) but not mutant enzyme ( lanes 10 , 15 , and 20 ). B , scanning densitometry for GFP expression quantitated by ImageJ is shown. GFP expression in the CMV empty vector alone ( lanes 1 , 6 , 11 , and 16 ) are normalized to 1, and expression in other lysates is relative to the respective control. Note that GFP expression is completely suppressed in the control sh cells transfected with Pax2/Grg4 in the presence or absence of PPM1B or mutant PPM1B (compare lane 2 with lanes 3–5 ). In the knock-out cells, co-expression of Pax2/Grg4 did not repress GFP expression (compare lanes 7 and 8 ; lanes 12 and 13 ; and lanes 17 and 18 ). Wild-type ( lanes 9 , 14 , and 19 ) but not mutant PPM1B ( lanes 10 , 15 , and 20 ) restores repression of GFP. All bars are averages of duplicates with error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p
    Figure Legend Snippet: PPM1B is required for Pax2- and Grg4-mediated gene repression. A , control negative sh GFP reporter cells or GFP reporter cells with shRNA 1, 2, or 4 were transfected with Grg4, Pax2, wild-type, or phosphatase-deficient PPM1B as indicated. Whole cell lysates were Western blotted ( WB ) for the indicated proteins with β-actin used as a loading control. Note that GFP expression increased upon Pax2 transfection ( lanes 2 , 7 , 12 , and 17 ) in all cell lines; this activation was inhibited by Grg4 ( lane 3 ) in control cells but not in PPM1B knockdown cells ( lanes 8 , 13 , and 18 ). The Grg4-mediated inhibition was rescued with wild-type PPM1B in the knockdown cells ( lanes 9 , 14 , and 19 ) but not mutant enzyme ( lanes 10 , 15 , and 20 ). B , scanning densitometry for GFP expression quantitated by ImageJ is shown. GFP expression in the CMV empty vector alone ( lanes 1 , 6 , 11 , and 16 ) are normalized to 1, and expression in other lysates is relative to the respective control. Note that GFP expression is completely suppressed in the control sh cells transfected with Pax2/Grg4 in the presence or absence of PPM1B or mutant PPM1B (compare lane 2 with lanes 3–5 ). In the knock-out cells, co-expression of Pax2/Grg4 did not repress GFP expression (compare lanes 7 and 8 ; lanes 12 and 13 ; and lanes 17 and 18 ). Wild-type ( lanes 9 , 14 , and 19 ) but not mutant PPM1B ( lanes 10 , 15 , and 20 ) restores repression of GFP. All bars are averages of duplicates with error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p

    Techniques Used: shRNA, Transfection, Western Blot, Expressing, Activation Assay, Inhibition, Mutagenesis, Plasmid Preparation, Knock-Out

    Pax2/Grg4-mediated chromatin remodeling at the Rap1A locus is PPM1B-dependent. A , GFP reporter cells with the control sh or cells with PPM1B shRNA 1 and shRNA 4 were transfected as noted and Western blotted ( WB ) for Grg4, PPM1B, and Pax2. Loading was normalized for β-galactosidase activity. B , RAP1A mRNA expression in the transfected cells from A . Rap1A expression in the CMV empty vector alone ( lanes 1 , 6 , and 11 ) are normalized to 1 and expression in other lanes are relative to the respective control. Note that in all cell lines, Pax2 stimulates Rap1a expression ( lanes 2 , 7 , and 12 ). In control cells, Pax2-Grg4 co-expression represses Rap1A expression ( lane 3 ) but not in the absence of PPM1B ( lanes 8 ,13). Rescue with wild-type PPM1B ( lanes 9 and 14 ) but not phosphatase-deficient PPM1B ( lanes 10 and 15 ) restores repression of Rap1A expression in the knock-out cells. All bars are averages of two experiments with PCR done in triplicate and error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p
    Figure Legend Snippet: Pax2/Grg4-mediated chromatin remodeling at the Rap1A locus is PPM1B-dependent. A , GFP reporter cells with the control sh or cells with PPM1B shRNA 1 and shRNA 4 were transfected as noted and Western blotted ( WB ) for Grg4, PPM1B, and Pax2. Loading was normalized for β-galactosidase activity. B , RAP1A mRNA expression in the transfected cells from A . Rap1A expression in the CMV empty vector alone ( lanes 1 , 6 , and 11 ) are normalized to 1 and expression in other lanes are relative to the respective control. Note that in all cell lines, Pax2 stimulates Rap1a expression ( lanes 2 , 7 , and 12 ). In control cells, Pax2-Grg4 co-expression represses Rap1A expression ( lane 3 ) but not in the absence of PPM1B ( lanes 8 ,13). Rescue with wild-type PPM1B ( lanes 9 and 14 ) but not phosphatase-deficient PPM1B ( lanes 10 and 15 ) restores repression of Rap1A expression in the knock-out cells. All bars are averages of two experiments with PCR done in triplicate and error bars indicating S.D. Note comparisons are made between all groups of transfections in each cell line, and significant comparisons are shown. *, p

    Techniques Used: shRNA, Transfection, Western Blot, Activity Assay, Expressing, Plasmid Preparation, Knock-Out, Polymerase Chain Reaction

    21) Product Images from "The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer"

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx727

    Expression of PRMT5 and WDR77 in breast cancer ( A ). Expression analysis of 109 paired samples from The Cancer Genome Atlas (TCGA) database shows significant overexpression of PRMT5 and WDR77 in breast cancer samples relative to matched normal samples ( B ). Fold expression of WDR77 and PRMT5 in breast normal (MCF10A), ER+ (MCF7, T47D) and ER- (MDA-MB-231, HCC38) breast cancer cell lines. Normalized to GAPDH ( C ). Immunoblotting of WDR77 and PRMT5 in nuclear and cytoplasmic extracts of breast normal and cancer cell lines. Actin was used as loading control. ( D ). Fold expression (relative to GAPDH) of WDR77 and PRMT5 and ( E ). Immunoblotting of the respective proteins in sh Scrambled, sh WDR77 and sh PRMT5-treated samples. Actin was used as loading control in E ( F ). (Top panel) Annexin V staining showing increased number of apoptotic cells upon loss of WDR77 and PRMT5 . (Bottom panel) Box plots of results from apoptosis assay for three biological replicates. Student's t-test * P
    Figure Legend Snippet: Expression of PRMT5 and WDR77 in breast cancer ( A ). Expression analysis of 109 paired samples from The Cancer Genome Atlas (TCGA) database shows significant overexpression of PRMT5 and WDR77 in breast cancer samples relative to matched normal samples ( B ). Fold expression of WDR77 and PRMT5 in breast normal (MCF10A), ER+ (MCF7, T47D) and ER- (MDA-MB-231, HCC38) breast cancer cell lines. Normalized to GAPDH ( C ). Immunoblotting of WDR77 and PRMT5 in nuclear and cytoplasmic extracts of breast normal and cancer cell lines. Actin was used as loading control. ( D ). Fold expression (relative to GAPDH) of WDR77 and PRMT5 and ( E ). Immunoblotting of the respective proteins in sh Scrambled, sh WDR77 and sh PRMT5-treated samples. Actin was used as loading control in E ( F ). (Top panel) Annexin V staining showing increased number of apoptotic cells upon loss of WDR77 and PRMT5 . (Bottom panel) Box plots of results from apoptosis assay for three biological replicates. Student's t-test * P

    Techniques Used: Expressing, Over Expression, Multiple Displacement Amplification, Staining, Apoptosis Assay

    Loss of PRMT5 and WDR77 leads to defects in alternative splicing and inclusion of A-T rich exons ( A and B ). (Top) Frequency of A or T upstream and downstream from splice sites of included exons (blue) excluded exons (green) and unaffected control exons (red). The dotted black line marks the meeting point of upstream and downstream datasets. (Below) Frequency of 5-base oligonucleotides in the regions around splice sites of included ( x -axis) versus control ( y -axis) exons in sh WDR77 (Left) and sh PRMT5 (Right) samples. Scatter plot of genes that are alternatively spliced and up- or downregulated upon loss of WDR77 ( C ) and PRMT5 ( D ) relative to scrambled shRNA control (Red- > 1.5-fold upregulated Blue- > 1.5-fold downregulated).
    Figure Legend Snippet: Loss of PRMT5 and WDR77 leads to defects in alternative splicing and inclusion of A-T rich exons ( A and B ). (Top) Frequency of A or T upstream and downstream from splice sites of included exons (blue) excluded exons (green) and unaffected control exons (red). The dotted black line marks the meeting point of upstream and downstream datasets. (Below) Frequency of 5-base oligonucleotides in the regions around splice sites of included ( x -axis) versus control ( y -axis) exons in sh WDR77 (Left) and sh PRMT5 (Right) samples. Scatter plot of genes that are alternatively spliced and up- or downregulated upon loss of WDR77 ( C ) and PRMT5 ( D ) relative to scrambled shRNA control (Red- > 1.5-fold upregulated Blue- > 1.5-fold downregulated).

    Techniques Used: shRNA

    Alternative splicing coupled mRNA decay of transcripts upon loss of PRMT5 . qPCR analysis showing (from left to right) relative levels of exon inclusion, pre-mRNA and the mRNA/pre-mRNA ratio in sh Scrambled and sh PRMT5 samples for ( A ) REPIN1/AP4 ( B ) ST3GAL6 ( C ) PFKM ( D ) TRNAU1AP/SECP43 . The illustrations depict the gene structure with exons shown as black boxes and introns as lines. The included exons are shown in red. Arrows indicate regions to which primers were designed. Student's t -test * P
    Figure Legend Snippet: Alternative splicing coupled mRNA decay of transcripts upon loss of PRMT5 . qPCR analysis showing (from left to right) relative levels of exon inclusion, pre-mRNA and the mRNA/pre-mRNA ratio in sh Scrambled and sh PRMT5 samples for ( A ) REPIN1/AP4 ( B ) ST3GAL6 ( C ) PFKM ( D ) TRNAU1AP/SECP43 . The illustrations depict the gene structure with exons shown as black boxes and introns as lines. The included exons are shown in red. Arrows indicate regions to which primers were designed. Student's t -test * P

    Techniques Used: Real-time Polymerase Chain Reaction

    ZNF326 is symmetrically dimethylated at R175 by the PRMT5/WDR77 complex ( A ). ZNF326 has two glycine-arginine rich motifs. Asterisk indicates R175 that was identified to be dimethylated ( B ). Immunoblots of immunoprecipitates showing symmetric dimethylation of ZNF326 ( C ). Representative tandem mass spectrum of the peptide GR(28.0314)GTPAYPESTFGSR {m/z:537.602 (+3). The fragment ion matching within 10 ppm are shown as either the B-ion (purple) or Y-ion (blue) series. The green dashed line indicates precursor m/z. The dotted lines in the fragmentation ladder sequence on the top the spectrum corresponds to the missing B-ion (purple) and Y-ion (blue) series ( D ). Mass Spectrometry analysis showing the relative abundance of random and dimethylated peptides in cells infected with sh Scrambled, sh WDR77 and sh PRMT5 and ( E ). Graphical representation of the same.
    Figure Legend Snippet: ZNF326 is symmetrically dimethylated at R175 by the PRMT5/WDR77 complex ( A ). ZNF326 has two glycine-arginine rich motifs. Asterisk indicates R175 that was identified to be dimethylated ( B ). Immunoblots of immunoprecipitates showing symmetric dimethylation of ZNF326 ( C ). Representative tandem mass spectrum of the peptide GR(28.0314)GTPAYPESTFGSR {m/z:537.602 (+3). The fragment ion matching within 10 ppm are shown as either the B-ion (purple) or Y-ion (blue) series. The green dashed line indicates precursor m/z. The dotted lines in the fragmentation ladder sequence on the top the spectrum corresponds to the missing B-ion (purple) and Y-ion (blue) series ( D ). Mass Spectrometry analysis showing the relative abundance of random and dimethylated peptides in cells infected with sh Scrambled, sh WDR77 and sh PRMT5 and ( E ). Graphical representation of the same.

    Techniques Used: Western Blot, Sequencing, Mass Spectrometry, Infection

    Networks of top interaction partners of WDR77 in the cytoplasm and nucleus identified by LC-MS/MS ( A ). Gene ontology analysis ( B ). Protein interaction network of top 15 interacting partners of WDR77 in the cytoplasm (red lines: newly identified interactions, black lines: previously characterized interactions) ( C ). Gene ontology analysis ( D ). Protein interaction network of top 15 interacting partners of WDR77 in the nucleus (red lines: newly identified interactions, black lines: previously characterized interactions) ( E ). Immunoblots of co-immunoprecipitations of WDR77, PRMT5 and ZNF326 (IP-Immunoprecipitation, WB-western blot).
    Figure Legend Snippet: Networks of top interaction partners of WDR77 in the cytoplasm and nucleus identified by LC-MS/MS ( A ). Gene ontology analysis ( B ). Protein interaction network of top 15 interacting partners of WDR77 in the cytoplasm (red lines: newly identified interactions, black lines: previously characterized interactions) ( C ). Gene ontology analysis ( D ). Protein interaction network of top 15 interacting partners of WDR77 in the nucleus (red lines: newly identified interactions, black lines: previously characterized interactions) ( E ). Immunoblots of co-immunoprecipitations of WDR77, PRMT5 and ZNF326 (IP-Immunoprecipitation, WB-western blot).

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Immunoprecipitation

    The PRMT5/WDR77 complex shapes the transcriptome of MDA-MB-231 cells through methylation of ZNF326. Methylation of ZNF326 by the PRMT5/WDR77 complex is essential for Pol II transcription across A-T rich genes. Loss of PRMT5 or WDR77 leads to a loss of methylation of ZNF326 that results in slow progression of Pol II causing the inclusion of A-T rich exons in target genes. A subset of these transcripts is targeted for degradation thereby altering the shape of the transcriptome of the cell. ( A ) Represents the influence of PRMT5/WDR77 to coordinate the rate at which transcription may help determine splicing patterns, where, the absence of PRMT5/WDR77 ( B ) effects the rate and aberrant inclusion of exons.
    Figure Legend Snippet: The PRMT5/WDR77 complex shapes the transcriptome of MDA-MB-231 cells through methylation of ZNF326. Methylation of ZNF326 by the PRMT5/WDR77 complex is essential for Pol II transcription across A-T rich genes. Loss of PRMT5 or WDR77 leads to a loss of methylation of ZNF326 that results in slow progression of Pol II causing the inclusion of A-T rich exons in target genes. A subset of these transcripts is targeted for degradation thereby altering the shape of the transcriptome of the cell. ( A ) Represents the influence of PRMT5/WDR77 to coordinate the rate at which transcription may help determine splicing patterns, where, the absence of PRMT5/WDR77 ( B ) effects the rate and aberrant inclusion of exons.

    Techniques Used: Multiple Displacement Amplification, Methylation

    22) Product Images from "Efficacy of Live-Attenuated H9N2 Influenza Vaccine Candidates Containing NS1 Truncations against H9N2 Avian Influenza Viruses"

    Article Title: Efficacy of Live-Attenuated H9N2 Influenza Vaccine Candidates Containing NS1 Truncations against H9N2 Avian Influenza Viruses

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.01086

    Detection of specific IgA and IgG level in mucosa. Chickens were immunized at 4 weeks of age, and lavage fluids from the trachea and nasal tissues were sampled at 7, 14, and 21 dpi. The antibody levels were detected using ELISA. All data are shown as the mean ± standard errors. Asterisks represent p -values
    Figure Legend Snippet: Detection of specific IgA and IgG level in mucosa. Chickens were immunized at 4 weeks of age, and lavage fluids from the trachea and nasal tissues were sampled at 7, 14, and 21 dpi. The antibody levels were detected using ELISA. All data are shown as the mean ± standard errors. Asterisks represent p -values

    Techniques Used: Enzyme-linked Immunosorbent Assay

    23) Product Images from "Global analysis of the nuclear processing of transcripts with unspliced U12-type introns by the exosome"

    Article Title: Global analysis of the nuclear processing of transcripts with unspliced U12-type introns by the exosome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku391

    U12-intron-containing transcripts are stabilized by the exosome knockdown. ( A ) Exon/intron structures of human VPS16 , MAPK12 and RCD8 genes and positions of the primers used in RT-PCR experiments (arrows). ( B ) Western blot illustrating the depletion of RRP41, DIS3, XRN2 and DCP2 upon siRNA-mediated knockdown. Cell lysates from cells treated with control siRNA were loaded in the indicated amounts. ( C ) RT-PCR analysis of the three transcripts described in panel (A) and beta-actin after knockdown of RRP41, DIS3, XRN2 or DCP2. The positions of the transcripts (i.e. mRNA versus pre-mRNA) are indicated on the left. U2 panels are shown in two versions, in the middle panel contrast settings are the same as in the U12 panel; in the rightmost panel the same gel is shown with darker settings to reveal the weak U2 intron signals. ( D ) Expression level of VSV-epitope-tagged RRP41 containing silent mutations at the siRNA target site, analyzed by western blotting using a polyclonal anti-RRP41 antibody and anti-VSV tag antibody. The relative intensity of the endogenous RRP41 signal (normalized to U1C signal) is shown below the panels. ( E ) Quantification of intron retention levels upon RRP41 knockdown and with or without RRP41 rescue. VPS16 , MAPK12 and RCD8 spliced and unspliced signals in agarose gels containing separated RT-PCR products were quantified (* P
    Figure Legend Snippet: U12-intron-containing transcripts are stabilized by the exosome knockdown. ( A ) Exon/intron structures of human VPS16 , MAPK12 and RCD8 genes and positions of the primers used in RT-PCR experiments (arrows). ( B ) Western blot illustrating the depletion of RRP41, DIS3, XRN2 and DCP2 upon siRNA-mediated knockdown. Cell lysates from cells treated with control siRNA were loaded in the indicated amounts. ( C ) RT-PCR analysis of the three transcripts described in panel (A) and beta-actin after knockdown of RRP41, DIS3, XRN2 or DCP2. The positions of the transcripts (i.e. mRNA versus pre-mRNA) are indicated on the left. U2 panels are shown in two versions, in the middle panel contrast settings are the same as in the U12 panel; in the rightmost panel the same gel is shown with darker settings to reveal the weak U2 intron signals. ( D ) Expression level of VSV-epitope-tagged RRP41 containing silent mutations at the siRNA target site, analyzed by western blotting using a polyclonal anti-RRP41 antibody and anti-VSV tag antibody. The relative intensity of the endogenous RRP41 signal (normalized to U1C signal) is shown below the panels. ( E ) Quantification of intron retention levels upon RRP41 knockdown and with or without RRP41 rescue. VPS16 , MAPK12 and RCD8 spliced and unspliced signals in agarose gels containing separated RT-PCR products were quantified (* P

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

    24) Product Images from "TNFSF14/LIGHT, a Non-Canonical NF-κB Stimulus, Induces the HIF Pathway"

    Article Title: TNFSF14/LIGHT, a Non-Canonical NF-κB Stimulus, Induces the HIF Pathway

    Journal: Cells

    doi: 10.3390/cells7080102

    LIGHT, a non-canonical NF-κB inducer, induces HIF expression and activity. ( A ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Whole cell lysates were prepared and analysed by western blot for the indicated non-canonical NF-κB pathway regulators, subunits and target genes. β-Actin was used as loading control. ( B ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior mRNA extraction and RT-qPCR analysis for RANTES transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated sample. The graphs depict mean and SEM determined from at least three independent experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( C ) HeLa cells, stably transfected with HRE luciferase reporter, were treated with 100 ng/mL LIGHT for 8 and 24 h prior to luciferase measurements. All of the values were normalised to the untreated sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. One way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( D ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior collection of whole cell lysates and western blot analysis for the depicted proteins. β-Actin was used as loading control. ( E ) HeLa cells were treated with 10 ng/mL TNF-α and 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for the depicted proteins. β-Actin was used as loading control. ( F ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for a subset of HIF-α specific targets. β-Actin was used as loading control. ( G ) HeLa cells and ( H ) A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h, prior mRNA extraction and RT-qPCR analysis for PHD2 gene transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated samples. The graph depicts mean and SEM determined from at least three independent biological experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( I ) Analysis of canonical NF-κB signalling following NIK expression vectors for 48 h prior to luciferase measurements. All the values were normalised to the control sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. Student t -test analysis was performed and significance determined as follows: ns = not significant, ** p ≤ 0.01. ( J ) HeLa cells were transfected with control and NIK expression vectors for 48 h prior to cell lysis. Western blot analysis was performed using the indicating antibodies. β-Actin was used as loading control.
    Figure Legend Snippet: LIGHT, a non-canonical NF-κB inducer, induces HIF expression and activity. ( A ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Whole cell lysates were prepared and analysed by western blot for the indicated non-canonical NF-κB pathway regulators, subunits and target genes. β-Actin was used as loading control. ( B ) HeLa cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior mRNA extraction and RT-qPCR analysis for RANTES transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated sample. The graphs depict mean and SEM determined from at least three independent experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( C ) HeLa cells, stably transfected with HRE luciferase reporter, were treated with 100 ng/mL LIGHT for 8 and 24 h prior to luciferase measurements. All of the values were normalised to the untreated sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. One way Anova analysis was performed and significance determined as follows: ns = not significant, *** p ≤ 0.001. ( D ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h prior collection of whole cell lysates and western blot analysis for the depicted proteins. β-Actin was used as loading control. ( E ) HeLa cells were treated with 10 ng/mL TNF-α and 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for the depicted proteins. β-Actin was used as loading control. ( F ) HeLa and A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h. Then, whole cell lysates were collected and western blot analysis was performed for a subset of HIF-α specific targets. β-Actin was used as loading control. ( G ) HeLa cells and ( H ) A549 cells were treated with 100 ng/mL LIGHT for 0, 4 and 24 h, prior mRNA extraction and RT-qPCR analysis for PHD2 gene transcript, normalised to Actin mRNA levels. All the values were normalised to the untreated samples. The graph depicts mean and SEM determined from at least three independent biological experiments. One-way Anova analysis was performed and significance determined as follows: ns = not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. ( I ) Analysis of canonical NF-κB signalling following NIK expression vectors for 48 h prior to luciferase measurements. All the values were normalised to the control sample. Graph depicts mean and SEM of a minimum of three independent biological experiments. Student t -test analysis was performed and significance determined as follows: ns = not significant, ** p ≤ 0.01. ( J ) HeLa cells were transfected with control and NIK expression vectors for 48 h prior to cell lysis. Western blot analysis was performed using the indicating antibodies. β-Actin was used as loading control.

    Techniques Used: Expressing, Activity Assay, Western Blot, Quantitative RT-PCR, Stable Transfection, Transfection, Luciferase, Lysis

    25) Product Images from "PSD-Zip70 Deficiency Causes Prefrontal Hypofunction Associated with Glutamatergic Synapse Maturation Defects by Dysregulation of Rap2 Activity"

    Article Title: PSD-Zip70 Deficiency Causes Prefrontal Hypofunction Associated with Glutamatergic Synapse Maturation Defects by Dysregulation of Rap2 Activity

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.2349-15.2015

    PSD-Zip70 interacts with both SPAR and PDZ-GEF1. a , HEK293T cells were transfected with Myc-tagged PSD-Zip70 in combination with HA-tagged PDZ-GEF1 or PDZ-GEF2, and co-IP assays were performed using cell lysates with an anti-Myc antibody. b , co-IP assay was performed using anti-PSD-Zip70, anti-SPAR, and anti-PDZ-GEF1 antibodies with lysates from mouse cortices. Endogenous SPAR and PDZ-GEF1 were co-immunoprecipitated with endogenous PSD-Zip70. The proteins in the immunoprecipitants were detected by Western blotting with the indicated antibodies. c , PSD-Zip70 was colocalized with SPAR (left) and PDZ-GEF1 (right) in cultured WT neurons. Scale bar, 10 μm. d , Schematic representation of the truncated fragments of PSD-Zip70. The N-terminal myristoylation motif, coiled-coil (coil) domain, leucine zipper (LZ) domain, and PDZ-binding motif are shown. Numbers are amino acid positions. e , f , HEK293T cells were transfected with Myc-tagged PSD-Zip70 fragments in combination with HA-PDZ-GEF1full, and co-IP assays were performed using cell lysates with anti-Myc ( e ) or anti-HA ( f ) antibodies. g , Schematic representation of the truncation series of PDZ-GEF1. The pseudocyclic-nucleotide binding domain (cNBD), Ras exchange motif (REM), PDZ domain, Ras-association (RA) domain, and RapGEF (GEF) domain are shown. h , HEK293T cells were transfected with various HA-tagged PDZ-GEF1 fragments in combination with Myc-tagged PSD-Zip70full, and co-IP assays were performed using the cell lysates with anti-Myc antibody. i , HEK293T cells were transfected with Myc-tagged PSD-Zip70 fragments in combination with HA-tagged PDZ-GEF1N(1–390), and co-IP assays were performed using cell lysates with anti-HA antibody. j , Schematic diagram of the interactions among PSD-Zip70, SPAR, and PDZ-GEF1. k , co-IP assay was performed in HEK293T cells with Myc-PSD-Zip70 in combination with HA-PDZ-GEF1 and/or FLAG-SPAR. The co-immunoprecipitated proteins with Myc-PSD-Zip70 were analyzed.
    Figure Legend Snippet: PSD-Zip70 interacts with both SPAR and PDZ-GEF1. a , HEK293T cells were transfected with Myc-tagged PSD-Zip70 in combination with HA-tagged PDZ-GEF1 or PDZ-GEF2, and co-IP assays were performed using cell lysates with an anti-Myc antibody. b , co-IP assay was performed using anti-PSD-Zip70, anti-SPAR, and anti-PDZ-GEF1 antibodies with lysates from mouse cortices. Endogenous SPAR and PDZ-GEF1 were co-immunoprecipitated with endogenous PSD-Zip70. The proteins in the immunoprecipitants were detected by Western blotting with the indicated antibodies. c , PSD-Zip70 was colocalized with SPAR (left) and PDZ-GEF1 (right) in cultured WT neurons. Scale bar, 10 μm. d , Schematic representation of the truncated fragments of PSD-Zip70. The N-terminal myristoylation motif, coiled-coil (coil) domain, leucine zipper (LZ) domain, and PDZ-binding motif are shown. Numbers are amino acid positions. e , f , HEK293T cells were transfected with Myc-tagged PSD-Zip70 fragments in combination with HA-PDZ-GEF1full, and co-IP assays were performed using cell lysates with anti-Myc ( e ) or anti-HA ( f ) antibodies. g , Schematic representation of the truncation series of PDZ-GEF1. The pseudocyclic-nucleotide binding domain (cNBD), Ras exchange motif (REM), PDZ domain, Ras-association (RA) domain, and RapGEF (GEF) domain are shown. h , HEK293T cells were transfected with various HA-tagged PDZ-GEF1 fragments in combination with Myc-tagged PSD-Zip70full, and co-IP assays were performed using the cell lysates with anti-Myc antibody. i , HEK293T cells were transfected with Myc-tagged PSD-Zip70 fragments in combination with HA-tagged PDZ-GEF1N(1–390), and co-IP assays were performed using cell lysates with anti-HA antibody. j , Schematic diagram of the interactions among PSD-Zip70, SPAR, and PDZ-GEF1. k , co-IP assay was performed in HEK293T cells with Myc-PSD-Zip70 in combination with HA-PDZ-GEF1 and/or FLAG-SPAR. The co-immunoprecipitated proteins with Myc-PSD-Zip70 were analyzed.

    Techniques Used: Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Cell Culture, Binding Assay

    PSD-Zip70 regulates spine morphology via modulation of the Rap2 activity. a , Typical spine morphologies of cultured neurons expressing EGFP with the indicated expression vectors. Forced expression of PDZ-GEF1, PDZ-GEF2, or an active form of Rap2 (Rap2V12) induced the formation of spines with smaller heads in WT neurons. Forced expression of an inactive form of Rap2 (Rap2N17), SPAR, or exogenous PSD-Zip70 recovered the spine head size in PSD-Zip70KO neurons. Scale bar, 5 μm. b , Quantification of protrusion width (trials ≥ 3, neurons ≥ 6, spines ≥ 235; F (8,25) = 68.85, p
    Figure Legend Snippet: PSD-Zip70 regulates spine morphology via modulation of the Rap2 activity. a , Typical spine morphologies of cultured neurons expressing EGFP with the indicated expression vectors. Forced expression of PDZ-GEF1, PDZ-GEF2, or an active form of Rap2 (Rap2V12) induced the formation of spines with smaller heads in WT neurons. Forced expression of an inactive form of Rap2 (Rap2N17), SPAR, or exogenous PSD-Zip70 recovered the spine head size in PSD-Zip70KO neurons. Scale bar, 5 μm. b , Quantification of protrusion width (trials ≥ 3, neurons ≥ 6, spines ≥ 235; F (8,25) = 68.85, p

    Techniques Used: Activity Assay, Cell Culture, Expressing

    PSD-Zip70 modulates Rap2 activity via SPAR- and PDZ-GEF1. a , Rap2 activity assay was performed in cultured WT neurons. Cultured neurons were cotransfected with exogenous FLAG-tagged Rap2 in combination with the indicated expression plasmids. Total and GTP-bound exogenous Rap2 were detected using anti-FLAG antibody. SPAR and PSD-Zip70 cooperatively suppressed the Rap2 activity. The RapGEF activity of PDZ-GEF1 and PDZ-GEF2 activated Rap2, and PSD-Zip70 coexpression suppressed the PDZ-GEF-dependent Rap2 activation. Quantified values are shown in the lower graph ( F (5,30) = 31.35, p
    Figure Legend Snippet: PSD-Zip70 modulates Rap2 activity via SPAR- and PDZ-GEF1. a , Rap2 activity assay was performed in cultured WT neurons. Cultured neurons were cotransfected with exogenous FLAG-tagged Rap2 in combination with the indicated expression plasmids. Total and GTP-bound exogenous Rap2 were detected using anti-FLAG antibody. SPAR and PSD-Zip70 cooperatively suppressed the Rap2 activity. The RapGEF activity of PDZ-GEF1 and PDZ-GEF2 activated Rap2, and PSD-Zip70 coexpression suppressed the PDZ-GEF-dependent Rap2 activation. Quantified values are shown in the lower graph ( F (5,30) = 31.35, p

    Techniques Used: Activity Assay, Cell Culture, Expressing, Activation Assay

    26) Product Images from "Targeting posttranslational modifications of RIOK1 inhibits the progression of colorectal and gastric cancers"

    Article Title: Targeting posttranslational modifications of RIOK1 inhibits the progression of colorectal and gastric cancers

    Journal: eLife

    doi: 10.7554/eLife.29511

    LSD1 interacts with RIOK1 endogenously. HCT116 cells were extracted and immunoprecipitated with an anti–LSD1 (left) or anti-RIOK1 (right) antibody. IP with rabbit IgG was used as the negative control. Western blot analysis was performed with the antibodies indicated.
    Figure Legend Snippet: LSD1 interacts with RIOK1 endogenously. HCT116 cells were extracted and immunoprecipitated with an anti–LSD1 (left) or anti-RIOK1 (right) antibody. IP with rabbit IgG was used as the negative control. Western blot analysis was performed with the antibodies indicated.

    Techniques Used: Immunoprecipitation, Negative Control, Western Blot

    Clinical relevance of RIOK1, SET7/9, LSD1, FBXO6 and CK2 Expression in patients with CRC. ( A ) Immunohistochemical staining of 104 human CRC for LSD1, CK2, SET7/9, and FBXO6 was performed. Representative photos of stains are shown in the groups with high (staining score, 5–8.0) and low (staining score, 0–4.0) expression of RIOK1. ( B ) Correlation between RIOK1 expression and LSD1, CK2, SET7/9, and FBXO6 expression in 104 clinical samples, respectively. ( C and D ) Kaplan–Meier plots of the overall survival and disease free survival in the patients ( n = 104) with CRC in the groups with high (staining score, 5–8.0) and low (staining score, 0–4.0) expression of LSD1, and CK2 and SET7/9 and FBXO6. ( E ) A proposed model illustrating that the RIOK1 methyl-phospho switch by SET7/9- CK2-LSD1 axis dictates the stability of RIOK1 and its role in CRC and GC growth and metastasis.
    Figure Legend Snippet: Clinical relevance of RIOK1, SET7/9, LSD1, FBXO6 and CK2 Expression in patients with CRC. ( A ) Immunohistochemical staining of 104 human CRC for LSD1, CK2, SET7/9, and FBXO6 was performed. Representative photos of stains are shown in the groups with high (staining score, 5–8.0) and low (staining score, 0–4.0) expression of RIOK1. ( B ) Correlation between RIOK1 expression and LSD1, CK2, SET7/9, and FBXO6 expression in 104 clinical samples, respectively. ( C and D ) Kaplan–Meier plots of the overall survival and disease free survival in the patients ( n = 104) with CRC in the groups with high (staining score, 5–8.0) and low (staining score, 0–4.0) expression of LSD1, and CK2 and SET7/9 and FBXO6. ( E ) A proposed model illustrating that the RIOK1 methyl-phospho switch by SET7/9- CK2-LSD1 axis dictates the stability of RIOK1 and its role in CRC and GC growth and metastasis.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    LSD1, CK2 and RIOK1 were in the same complex. Two-step co-IP assay. The HCT116 cells were transfected with the HA-LSD1, Flag-RIOK1 and Myc-CK2 plasmids as indicated. Cells were lysed. The first immunoprecipitation was performed with an anti-Flag antibody. The complex was eluted with the Flag peptide, followed by the second step of immunoprecipitation with an anti-HA antibody. Protein samples from each step were subjected to western blot analysis.
    Figure Legend Snippet: LSD1, CK2 and RIOK1 were in the same complex. Two-step co-IP assay. The HCT116 cells were transfected with the HA-LSD1, Flag-RIOK1 and Myc-CK2 plasmids as indicated. Cells were lysed. The first immunoprecipitation was performed with an anti-Flag antibody. The complex was eluted with the Flag peptide, followed by the second step of immunoprecipitation with an anti-HA antibody. Protein samples from each step were subjected to western blot analysis.

    Techniques Used: Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

    27) Product Images from "Rab22A recruits BLOC‐1 and BLOC‐2 to promote the biogenesis of recycling endosomes"

    Article Title: Rab22A recruits BLOC‐1 and BLOC‐2 to promote the biogenesis of recycling endosomes

    Journal: EMBO Reports

    doi: 10.15252/embr.201845918

    Rab22A regulates membrane association of BLOC ‐1 and BLOC ‐2, and forms a complex with BLOC ‐1‐ BLOC ‐2‐ KIF 13A Subcellular membrane fractionation of control and Rab22A‐, BLOC‐1‐, BLOC‐2‐knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red coloured box emphasizes BLOC‐1 membrane association in the respective cell types. Membrane‐cytosol fractionation of homogenates from HeLa cells (B) or melanocytes (C) as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels. Pull‐down of His‐Rab22A WT using HeLa (D) or melanocyte (E) lysate. In (E), the beads were preloaded with GTPγS or GDP. Pull‐down of different His‐KIF13A domains using HeLa cell lysate. Model illustrating the recruitment (left) and association (middle) of Rab22A, BLOC‐1 and BLOC‐2 in a sequential manner onto the membrane buds of E/SEs followed by interaction with KIF13A motor. Rab22A‐BLOC‐1‐BLOC‐2 complex possibly extends the membrane buds into RE tubules with KIF13A motor (right) movement on microtubules. Data information: In (A–F), * indicates non‐specific bands. In (D–F), the bead‐bound His‐Rab22A/His‐KIF13A domains were shown on the Coomassie‐stained gels separately or in Fig 1 F.
    Figure Legend Snippet: Rab22A regulates membrane association of BLOC ‐1 and BLOC ‐2, and forms a complex with BLOC ‐1‐ BLOC ‐2‐ KIF 13A Subcellular membrane fractionation of control and Rab22A‐, BLOC‐1‐, BLOC‐2‐knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red coloured box emphasizes BLOC‐1 membrane association in the respective cell types. Membrane‐cytosol fractionation of homogenates from HeLa cells (B) or melanocytes (C) as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels. Pull‐down of His‐Rab22A WT using HeLa (D) or melanocyte (E) lysate. In (E), the beads were preloaded with GTPγS or GDP. Pull‐down of different His‐KIF13A domains using HeLa cell lysate. Model illustrating the recruitment (left) and association (middle) of Rab22A, BLOC‐1 and BLOC‐2 in a sequential manner onto the membrane buds of E/SEs followed by interaction with KIF13A motor. Rab22A‐BLOC‐1‐BLOC‐2 complex possibly extends the membrane buds into RE tubules with KIF13A motor (right) movement on microtubules. Data information: In (A–F), * indicates non‐specific bands. In (D–F), the bead‐bound His‐Rab22A/His‐KIF13A domains were shown on the Coomassie‐stained gels separately or in Fig 1 F.

    Techniques Used: Fractionation, Staining

    Analysis of cargo recycling in control and Rab22A‐, BLOC ‐1‐, BLOC ‐2‐knockdown HeLa cells IFM images of KIF13A‐YFP‐transfected control and knockdown HeLa cells as indicated. Arrowheads point to the localization of KIF13A to E/SEs or REs. Membrane‐cytosol fractionation of HeLa cell homogenate for the localization of KIF13A. *, non‐specific bands. IFM images of control and Rab22A‐, BLOC‐1‐knockdown HeLa cells that were stained with LAMP‐2 or internalized with fluorescein–dextran. Cell surface levels of LAMP‐1 and M6PR in control and Rab22A‐, BLOC‐1‐knockdown HeLa cells measured using flow cytometry. Normalized mean fluorescence intensity (MFI) was calculated (mean ± SEM) and then plotted. n = 3. IFM images of HeLa cells that were subjected to Tf‐Alexa Fluor 594 recycling kinetics. Fluorescence intensities Tf in the images of (E) were quantified and plotted (mean ± SEM). n = 3. n c = total number of cells. Immunoblotting analysis of Tf receptor in HeLa cells as indicated. Protein band intensities were quantified and indicated on the gels. Data information: In (A, C, E), arrows point to the localization of cytoskeletal proteins (A) or internalized dextran or lysosomes (C) or accumulation of Tf to the intracellular vesicles (E). Scale bars: 10 μm. In (D, F), * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns = not significant (unpaired Student's t ‐test).
    Figure Legend Snippet: Analysis of cargo recycling in control and Rab22A‐, BLOC ‐1‐, BLOC ‐2‐knockdown HeLa cells IFM images of KIF13A‐YFP‐transfected control and knockdown HeLa cells as indicated. Arrowheads point to the localization of KIF13A to E/SEs or REs. Membrane‐cytosol fractionation of HeLa cell homogenate for the localization of KIF13A. *, non‐specific bands. IFM images of control and Rab22A‐, BLOC‐1‐knockdown HeLa cells that were stained with LAMP‐2 or internalized with fluorescein–dextran. Cell surface levels of LAMP‐1 and M6PR in control and Rab22A‐, BLOC‐1‐knockdown HeLa cells measured using flow cytometry. Normalized mean fluorescence intensity (MFI) was calculated (mean ± SEM) and then plotted. n = 3. IFM images of HeLa cells that were subjected to Tf‐Alexa Fluor 594 recycling kinetics. Fluorescence intensities Tf in the images of (E) were quantified and plotted (mean ± SEM). n = 3. n c = total number of cells. Immunoblotting analysis of Tf receptor in HeLa cells as indicated. Protein band intensities were quantified and indicated on the gels. Data information: In (A, C, E), arrows point to the localization of cytoskeletal proteins (A) or internalized dextran or lysosomes (C) or accumulation of Tf to the intracellular vesicles (E). Scale bars: 10 μm. In (D, F), * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns = not significant (unpaired Student's t ‐test).

    Techniques Used: Transfection, Fractionation, Staining, Flow Cytometry, Cytometry, Fluorescence

    Analysis of endosomal Rab RNA i screen and the localization of Rabs to RE s IFM images of HeLa cells to study the localization of KIF13A‐YFP to REs. PCR analysis of Rab‐knockdown HeLa cells to measure the knockdown efficiency of each shRNA as indicated. DNA band intensities were quantified and indicated on the gels. Subcellular fractionation of HeLa cells to study the localization of different Rabs (red box) with respect to STX13 or AP‐1. IFM analysis of HeLa cells that were transfected with different constructs as indicated. Live cell imaging of mCherry‐Rab22A with respect to GFP‐Rab9A in HeLa cells. Arrowheads point to the Rab22A‐positive tubular structures. Magnified view of insets (at 0, 16, 40 s) are shown separately. Scale bars: 10 μm. IFM images of Rab22A sh, BLOC‐1 sh and control sh HeLa cells that were transfected with indicated constructs. Graphs represent the measurement of corrected total cell fluorescence (CTCF) in HeLa cells of Fig 2 A. n = 6–8 cells. Average CTCF values (AU: arbitrary units) and their respective fold changes (mean ± SEM) are indicated. Data information: In (A), arrowheads and arrows point to the KIF13A‐positive tubular REs and E/SEs, respectively. In (D, E, G), arrowheads point to the STX13‐ or KIF13A‐positive tubular REs or E/SEs. Scale bars: 10 μm.
    Figure Legend Snippet: Analysis of endosomal Rab RNA i screen and the localization of Rabs to RE s IFM images of HeLa cells to study the localization of KIF13A‐YFP to REs. PCR analysis of Rab‐knockdown HeLa cells to measure the knockdown efficiency of each shRNA as indicated. DNA band intensities were quantified and indicated on the gels. Subcellular fractionation of HeLa cells to study the localization of different Rabs (red box) with respect to STX13 or AP‐1. IFM analysis of HeLa cells that were transfected with different constructs as indicated. Live cell imaging of mCherry‐Rab22A with respect to GFP‐Rab9A in HeLa cells. Arrowheads point to the Rab22A‐positive tubular structures. Magnified view of insets (at 0, 16, 40 s) are shown separately. Scale bars: 10 μm. IFM images of Rab22A sh, BLOC‐1 sh and control sh HeLa cells that were transfected with indicated constructs. Graphs represent the measurement of corrected total cell fluorescence (CTCF) in HeLa cells of Fig 2 A. n = 6–8 cells. Average CTCF values (AU: arbitrary units) and their respective fold changes (mean ± SEM) are indicated. Data information: In (A), arrowheads and arrows point to the KIF13A‐positive tubular REs and E/SEs, respectively. In (D, E, G), arrowheads point to the STX13‐ or KIF13A‐positive tubular REs or E/SEs. Scale bars: 10 μm.

    Techniques Used: Polymerase Chain Reaction, shRNA, Fractionation, Transfection, Construct, Live Cell Imaging, Fluorescence

    Selected endosomal Rab RNA i screen identified Rab22A as a regulator of RE dynamics IFM images of KIF13A‐YFP‐transfected control and Rab‐knockdown HeLa cells. T N : average tubule number (mean ± SEM, n = 3). Graphs represent the measurement of KIF13A‐positive T N (B) and T L (C) in HeLa cells of Fig 1 A (mean ± SEM). n = 3. n c : total number of cells. n t : total number of tubules. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns = not significant (unpaired Student's t ‐test). IFM images of KIF13A‐YFP and mCherry‐Rab7A/11A/22A‐transfected HeLa cells. Live cell imaging of GFP/mCherry‐Rab22A with respect to mCherry‐Rab11A or GFP‐Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 s) are shown separately. Pull‐down of different His‐KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead‐bound His‐KIF13A in each pull‐down was shown on the Coomassie‐stained gel. *, non‐specific bands. Note, part of this experiment was shown in Fig 5 F. Data information: In (A, D, E), arrowheads and arrows point to the KIF13A‐/Rab22A‐positive tubular REs and E/SEs, respectively. Scale bars: 10 μm.
    Figure Legend Snippet: Selected endosomal Rab RNA i screen identified Rab22A as a regulator of RE dynamics IFM images of KIF13A‐YFP‐transfected control and Rab‐knockdown HeLa cells. T N : average tubule number (mean ± SEM, n = 3). Graphs represent the measurement of KIF13A‐positive T N (B) and T L (C) in HeLa cells of Fig 1 A (mean ± SEM). n = 3. n c : total number of cells. n t : total number of tubules. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and ns = not significant (unpaired Student's t ‐test). IFM images of KIF13A‐YFP and mCherry‐Rab7A/11A/22A‐transfected HeLa cells. Live cell imaging of GFP/mCherry‐Rab22A with respect to mCherry‐Rab11A or GFP‐Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 s) are shown separately. Pull‐down of different His‐KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead‐bound His‐KIF13A in each pull‐down was shown on the Coomassie‐stained gel. *, non‐specific bands. Note, part of this experiment was shown in Fig 5 F. Data information: In (A, D, E), arrowheads and arrows point to the KIF13A‐/Rab22A‐positive tubular REs and E/SEs, respectively. Scale bars: 10 μm.

    Techniques Used: Transfection, Live Cell Imaging, Staining

    Rab22A localizes to the E/ SE and RE s and regulates RE dynamics IFM images of KIF13A‐YFP and mCherry‐Rab22A WT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A‐positive tubular REs and E/SEs, respectively. The colocalization coefficient ( r , in mean ± SEM) between two markers was indicated separately. Scale bars: 10 μm. Graphs represent the measurement of KIF13A‐positive T N (B) and T L (C) in HeLa cells of Fig 2 A (mean ± SEM). n = 3. n c : total number of cells. n t : total number of tubules. ** P ≤ 0.01, *** P ≤ 0.001 and ns = not significant (unpaired Student's t ‐test). Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle‐specific proteins. *, non‐specific bands.
    Figure Legend Snippet: Rab22A localizes to the E/ SE and RE s and regulates RE dynamics IFM images of KIF13A‐YFP and mCherry‐Rab22A WT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A‐positive tubular REs and E/SEs, respectively. The colocalization coefficient ( r , in mean ± SEM) between two markers was indicated separately. Scale bars: 10 μm. Graphs represent the measurement of KIF13A‐positive T N (B) and T L (C) in HeLa cells of Fig 2 A (mean ± SEM). n = 3. n c : total number of cells. n t : total number of tubules. ** P ≤ 0.01, *** P ≤ 0.001 and ns = not significant (unpaired Student's t ‐test). Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle‐specific proteins. *, non‐specific bands.

    Techniques Used: Fractionation

    Rab22A functions upstream in the pathway of BLOC ‐1 and BLOC ‐2 and regulates RE dynamics IFM images of KIF13A‐YFP‐transfected control and Rab22A‐, BLOC‐1 Mu ‐, BLOC‐2 HPS6 ‐knockdown HeLa cells. Cells were stained for AP‐1 (γ) or internalized with Tf‐Alexa Fluor 594. Graphs represent the measurement of KIF13A‐positive T N (B) and T L (C) in HeLa cells of Fig 3 A (mean ± SEM). n = 3. n c : total number of cells. n t : total number of tubules. * P ≤ 0.05 and *** P ≤ 0.001 (unpaired Student's t ‐test). Immunoblotting analysis of proteins in control and Rab22A‐, BLOC‐1‐, BLOC‐2‐depleted HeLa cells. *, non‐specific bands. Protein band intensities were quantified and indicated on the gels. IFM images of mCherry‐Rab22A and KIF13A‐YFP cotransfected control and BLOC‐1 Mu ‐, BLOC‐2 HPS6 ‐knockdown HeLa cells. Data information: In (A, E), arrowheads and arrows point to the KIF13A‐/Rab22A‐positive tubular REs and E/SEs, respectively. Scale bars: 10 μm.
    Figure Legend Snippet: Rab22A functions upstream in the pathway of BLOC ‐1 and BLOC ‐2 and regulates RE dynamics IFM images of KIF13A‐YFP‐transfected control and Rab22A‐, BLOC‐1 Mu ‐, BLOC‐2 HPS6 ‐knockdown HeLa cells. Cells were stained for AP‐1 (γ) or internalized with Tf‐Alexa Fluor 594. Graphs represent the measurement of KIF13A‐positive T N (B) and T L (C) in HeLa cells of Fig 3 A (mean ± SEM). n = 3. n c : total number of cells. n t : total number of tubules. * P ≤ 0.05 and *** P ≤ 0.001 (unpaired Student's t ‐test). Immunoblotting analysis of proteins in control and Rab22A‐, BLOC‐1‐, BLOC‐2‐depleted HeLa cells. *, non‐specific bands. Protein band intensities were quantified and indicated on the gels. IFM images of mCherry‐Rab22A and KIF13A‐YFP cotransfected control and BLOC‐1 Mu ‐, BLOC‐2 HPS6 ‐knockdown HeLa cells. Data information: In (A, E), arrowheads and arrows point to the KIF13A‐/Rab22A‐positive tubular REs and E/SEs, respectively. Scale bars: 10 μm.

    Techniques Used: Transfection, Staining

    28) Product Images from "The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted"

    Article Title: The ATR-mediated S phase checkpoint prevents rereplication in mammalian cells when licensing control is disrupted

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200704138

    RPA2, MCM2, and Rb are important effector proteins downstream of ATR to inhibit Cdt1-induced rereplication. (A) 48 h after Ad-vec or Ad-Cdt1 infection, chromatin was purified from U2OS cells expressing shRNA-ATR, shRNA-ATM, or vector MKO (left), or cell lysates were prepared from U2OS or IMR90 cells (right). The adenovirus titers U2OS (5 × 10 7 pfu/ml, MOI = 25) and IMR90 (6 × 10 8 pfu/ml, MOI = 600) were used. After 1 h, 50 J/m 2 UV treatment was used as a positive control for MCM2 phosphorylation. Western blot analyses were performed as indicated. RPA2p, phosphorylated RPA2; MCM2-pS108, phosphorylated MCM2 at S108. (B) U2OS cell lines expressing the HA-RPA2 wild type or HA-RPA2-phospho mutant (S4A/S8A/S11A/S12A/S13A/T21A/S33A) with endogenous RPA2 silenced by shRNA were generated as described previously ( Olson et al., 2006 ). FACS analyses were performed 48 h after Ad-vec or Ad-Cdt1 infection (5 × 10 7 pfu/ml, MOI = 25). Western blot analyses were performed as indicated. endo-RPA2, endogenous RPA2; HA-RPA2p, phosphorylated HA-tagged RPA2. (C) U2OS cells carrying tetracycline-regulated constitutively active Rb were infected with Ad-vec or Ad-Cdt1 (5 × 10 7 pfu/ml, MOI = 25) in the presence of tetracycline (+tet, un-Rb off) or 24 h after removal of tetracycline (−tet, un-Rb on). 36 h after infection, cell cycle profiles were monitored by FACS analysis (top). Rb was silenced in A549 and T98G cells by expressing shRNA from retroviral vector MKO. FACS analysis was performed 48 h after Ad-vec or Ad-Cdt1 infection (bottom; A549: 6 × 10 8 pfu/ml, MOI = 400; T98G: 6 × 10 8 pfu/ml, MOI = 460). (D) Cell lysates were prepared from IMR90 or A549 cells expressing ATR-shRNA or vector MKO 20 or 40 h after Ad-vec or Ad-Cdt1 infection (6 × 10 8 pfu/ml; IMR90, MOI = 600; A549, MOI = 400). Rb phosphorylation was analyzed by the antibody G99-549, which specifically recognizes nonphosphorylated S608 species. Ku80 was used as a loading control.
    Figure Legend Snippet: RPA2, MCM2, and Rb are important effector proteins downstream of ATR to inhibit Cdt1-induced rereplication. (A) 48 h after Ad-vec or Ad-Cdt1 infection, chromatin was purified from U2OS cells expressing shRNA-ATR, shRNA-ATM, or vector MKO (left), or cell lysates were prepared from U2OS or IMR90 cells (right). The adenovirus titers U2OS (5 × 10 7 pfu/ml, MOI = 25) and IMR90 (6 × 10 8 pfu/ml, MOI = 600) were used. After 1 h, 50 J/m 2 UV treatment was used as a positive control for MCM2 phosphorylation. Western blot analyses were performed as indicated. RPA2p, phosphorylated RPA2; MCM2-pS108, phosphorylated MCM2 at S108. (B) U2OS cell lines expressing the HA-RPA2 wild type or HA-RPA2-phospho mutant (S4A/S8A/S11A/S12A/S13A/T21A/S33A) with endogenous RPA2 silenced by shRNA were generated as described previously ( Olson et al., 2006 ). FACS analyses were performed 48 h after Ad-vec or Ad-Cdt1 infection (5 × 10 7 pfu/ml, MOI = 25). Western blot analyses were performed as indicated. endo-RPA2, endogenous RPA2; HA-RPA2p, phosphorylated HA-tagged RPA2. (C) U2OS cells carrying tetracycline-regulated constitutively active Rb were infected with Ad-vec or Ad-Cdt1 (5 × 10 7 pfu/ml, MOI = 25) in the presence of tetracycline (+tet, un-Rb off) or 24 h after removal of tetracycline (−tet, un-Rb on). 36 h after infection, cell cycle profiles were monitored by FACS analysis (top). Rb was silenced in A549 and T98G cells by expressing shRNA from retroviral vector MKO. FACS analysis was performed 48 h after Ad-vec or Ad-Cdt1 infection (bottom; A549: 6 × 10 8 pfu/ml, MOI = 400; T98G: 6 × 10 8 pfu/ml, MOI = 460). (D) Cell lysates were prepared from IMR90 or A549 cells expressing ATR-shRNA or vector MKO 20 or 40 h after Ad-vec or Ad-Cdt1 infection (6 × 10 8 pfu/ml; IMR90, MOI = 600; A549, MOI = 400). Rb phosphorylation was analyzed by the antibody G99-549, which specifically recognizes nonphosphorylated S608 species. Ku80 was used as a loading control.

    Techniques Used: Infection, Purification, Expressing, shRNA, Plasmid Preparation, Positive Control, Western Blot, Mutagenesis, Generated, FACS

    29) Product Images from "Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells"

    Article Title: Inhibition of SNW1 association with spliceosomal proteins promotes apoptosis in breast cancer cells

    Journal: Cancer Medicine

    doi: 10.1002/cam4.366

    The SKIP domain of SNW1 associates with EFTUD2 and SNRNP200. (A) Flag-SNW1- or Flag-expressing 293T cells were lysed and immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. (B) Flag-SNW1 was expressed in 293T cells together with either GFP-EFTUD2, GFP-SNRNP200 or GFP-PRPF8, and after 24 h, cells were lysed and immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with an anti-GFP or anti-Flag antibody. (C) Schematic representation of the deletion constructs of SNW1. (D) GFP-EFTUD2 or GFP-SNRNP200 was expressed in 293T cells together with deletion mutants of Flag-tagged SNW1. After 24 h, the cells were lysed and immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were immunoblotted with an anti-GFP or anti-Flag antibody. The arrows indicate the heavy chain and light chain of the antibody.
    Figure Legend Snippet: The SKIP domain of SNW1 associates with EFTUD2 and SNRNP200. (A) Flag-SNW1- or Flag-expressing 293T cells were lysed and immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. (B) Flag-SNW1 was expressed in 293T cells together with either GFP-EFTUD2, GFP-SNRNP200 or GFP-PRPF8, and after 24 h, cells were lysed and immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with an anti-GFP or anti-Flag antibody. (C) Schematic representation of the deletion constructs of SNW1. (D) GFP-EFTUD2 or GFP-SNRNP200 was expressed in 293T cells together with deletion mutants of Flag-tagged SNW1. After 24 h, the cells were lysed and immunoprecipitated with an anti-Flag antibody. The immunoprecipitates were immunoblotted with an anti-GFP or anti-Flag antibody. The arrows indicate the heavy chain and light chain of the antibody.

    Techniques Used: Expressing, Immunoprecipitation, Construct

    The N-terminus of SNW1 and the C-terminus of SNRNP200 directly associate with the SKIP domain of SNW1. (A) The interaction of Flag-SNW1 with full-length as well as deletion mutants of GFP-tagged EFTUD2 in 293T cells was examined by immunoprecipitation with an anti-Flag antibody. (B) The interaction of GFP-SNRNP200 with Flag-tagged full-length or 1-260 EFTUD2 in 293T cells was examined by immunoprecipitation with an anti-Flag antibody. The arrow indicates the heavy chain of the antibody. (C) In vitro-translated HA-tagged EFTUD2-1-260 was mixed with GST or GST-fused SNW1-174-335 bound to glutathione agarose beads and affinity precipitated. The precipitates were immunoblotted with the indicated antibodies. (D) The interaction of Flag-SNW1 with GFP-tagged deletion mutants of SNRNP200 in 293T cells was examined by immunoprecipitation with an anti-Flag antibody. (E) In vitro-translated HA-tagged deletion mutants of SNRNP200 were mixed with GST or GST-fused SNW1-174-335 bound to glutathione agarose beads and affinity precipitated. The precipitates were immunoblotted with the indicated antibodies. (F) Schematic representation of SNW1 association with EFTUD2 and SNRNP200.
    Figure Legend Snippet: The N-terminus of SNW1 and the C-terminus of SNRNP200 directly associate with the SKIP domain of SNW1. (A) The interaction of Flag-SNW1 with full-length as well as deletion mutants of GFP-tagged EFTUD2 in 293T cells was examined by immunoprecipitation with an anti-Flag antibody. (B) The interaction of GFP-SNRNP200 with Flag-tagged full-length or 1-260 EFTUD2 in 293T cells was examined by immunoprecipitation with an anti-Flag antibody. The arrow indicates the heavy chain of the antibody. (C) In vitro-translated HA-tagged EFTUD2-1-260 was mixed with GST or GST-fused SNW1-174-335 bound to glutathione agarose beads and affinity precipitated. The precipitates were immunoblotted with the indicated antibodies. (D) The interaction of Flag-SNW1 with GFP-tagged deletion mutants of SNRNP200 in 293T cells was examined by immunoprecipitation with an anti-Flag antibody. (E) In vitro-translated HA-tagged deletion mutants of SNRNP200 were mixed with GST or GST-fused SNW1-174-335 bound to glutathione agarose beads and affinity precipitated. The precipitates were immunoblotted with the indicated antibodies. (F) Schematic representation of SNW1 association with EFTUD2 and SNRNP200.

    Techniques Used: Immunoprecipitation, In Vitro

    30) Product Images from "ARID3B Induces Tumor Necrosis Factor Alpha Mediated Apoptosis While a Novel ARID3B Splice Form Does Not Induce Cell Death"

    Article Title: ARID3B Induces Tumor Necrosis Factor Alpha Mediated Apoptosis While a Novel ARID3B Splice Form Does Not Induce Cell Death

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0042159

    EGFR modestly regulates the expression of ARID3B splice forms. QRT-PCR for ARID3B Fl and ARID3B Sh was performed on total RNA isolated from OVCA 429, OVCA 433 and DOV13 ovarian cancer cells grown in 10% serum conditions (FS) ( A and C ) or serum starved (SS) ( B and D ) for 24 h and treated with or without 20 nM of EGF or 10 nM of PDGF for an additional 24 h. QRT-PCR was performed for ARID3B Fl ( A and B ) and ARID3B Sh ( C and D ). Expression was normalized to 18 s rRNA. Fold expression was normalized to non-EGF treated cells (SS or FS) and was expressed as the mean ± SEM of triplicate measurements. Statistical analysis was performed to determine if there were any significant changes between FS vs. treatment with FS and EGF in all three ovarian cancer cell lines and for both ARID3B isoforms ( A and C ). We also performed statistical analysis to determine if there were any significant changes between SS vs. treatment with SS and FS or PDGF or EGF in all three ovarian cancer cell lines and for both ARID3B isoforms ( B and D ). E . OVCA 429 and OVCA 433 ovarian cancer cells grown in serum starved (SS) or 10% serum conditions (FS) for 24 h and treated with or without 20 nM of EGF for an additional 24 h. Whole cell lysates were analyzed by western blot using anti-ARID3B and anti-GAPDH (control) antibodies. Results were normalized to GADPH expression and compared to the expression of cell not treated with EGF (SS or FS). The densitometry evaluation of the western blot analyses for ARID3B Fl and ARID3B Sh in ovarian cancer cells were analyzed (value under blot). Statistical analysis was performed to determine if there were any significant changes between (FS) vs. (FS and EGF) and (SS) vs. (SS and EGF) in all three ovarian cancer cell lines and for both ARID3B isoforms fold expression was expressed as the mean of triplicate measurements. [*p
    Figure Legend Snippet: EGFR modestly regulates the expression of ARID3B splice forms. QRT-PCR for ARID3B Fl and ARID3B Sh was performed on total RNA isolated from OVCA 429, OVCA 433 and DOV13 ovarian cancer cells grown in 10% serum conditions (FS) ( A and C ) or serum starved (SS) ( B and D ) for 24 h and treated with or without 20 nM of EGF or 10 nM of PDGF for an additional 24 h. QRT-PCR was performed for ARID3B Fl ( A and B ) and ARID3B Sh ( C and D ). Expression was normalized to 18 s rRNA. Fold expression was normalized to non-EGF treated cells (SS or FS) and was expressed as the mean ± SEM of triplicate measurements. Statistical analysis was performed to determine if there were any significant changes between FS vs. treatment with FS and EGF in all three ovarian cancer cell lines and for both ARID3B isoforms ( A and C ). We also performed statistical analysis to determine if there were any significant changes between SS vs. treatment with SS and FS or PDGF or EGF in all three ovarian cancer cell lines and for both ARID3B isoforms ( B and D ). E . OVCA 429 and OVCA 433 ovarian cancer cells grown in serum starved (SS) or 10% serum conditions (FS) for 24 h and treated with or without 20 nM of EGF for an additional 24 h. Whole cell lysates were analyzed by western blot using anti-ARID3B and anti-GAPDH (control) antibodies. Results were normalized to GADPH expression and compared to the expression of cell not treated with EGF (SS or FS). The densitometry evaluation of the western blot analyses for ARID3B Fl and ARID3B Sh in ovarian cancer cells were analyzed (value under blot). Statistical analysis was performed to determine if there were any significant changes between (FS) vs. (FS and EGF) and (SS) vs. (SS and EGF) in all three ovarian cancer cell lines and for both ARID3B isoforms fold expression was expressed as the mean of triplicate measurements. [*p

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Western Blot

    Subcellular localization of ARID3B splice forms. Parental OVCA 429 cells ( A ) or OVCA 429 cells transduced with GFP ( B ), ARID3B Fl ( C ) or ARID3B Sh ( D ) were serum starved (SS) for 24 h, treated with or without 20 nM EGF for 24 h, and fractionated into cytoplasmic, membrane, nuclear and chromatin-bound extract fractions. Western blot was performed for ARID3B, Tom 20, Histone H3 and Lamin B1. Cytoplasmic fraction [lanes 1 (no EGF) and 2 (20 nM EGF)], membrane fraction [lanes 3 (no EGF) and 4 (20 nM EGF)], nuclear soluble fraction [lanes 5 (no EGF) and 6 (20 nM EGF)] and chromatin-bound fraction [lanes 7 (no EGF) and 8 (20 nM EGF)].
    Figure Legend Snippet: Subcellular localization of ARID3B splice forms. Parental OVCA 429 cells ( A ) or OVCA 429 cells transduced with GFP ( B ), ARID3B Fl ( C ) or ARID3B Sh ( D ) were serum starved (SS) for 24 h, treated with or without 20 nM EGF for 24 h, and fractionated into cytoplasmic, membrane, nuclear and chromatin-bound extract fractions. Western blot was performed for ARID3B, Tom 20, Histone H3 and Lamin B1. Cytoplasmic fraction [lanes 1 (no EGF) and 2 (20 nM EGF)], membrane fraction [lanes 3 (no EGF) and 4 (20 nM EGF)], nuclear soluble fraction [lanes 5 (no EGF) and 6 (20 nM EGF)] and chromatin-bound fraction [lanes 7 (no EGF) and 8 (20 nM EGF)].

    Techniques Used: Transduction, Western Blot

    ARID3B Fl induces apoptosis in ovarian carcinoma cells via TNFα/TRAIL pathways. Western blot using anti-Caspase 7, anti-Caspase 10, anti-TNF-R2, anti-TRAIL, anti-TRADD and anti- GAPDH (control) antibodies on transduced OVCA 429 ( A ) and OVCA 433 ( B ) cells. The densitometry evaluations of the western blots were analyzed (value under blot). Results were normalized to GAPDH expression and compared to parental ovarian cancer cells. Statistical analysis was performed to determine if there were any significant changes between parental ovarian cancer cells vs. GFP, ARID3B Sh or ARID3B Fl transduced ovarian cancer cells. [*p
    Figure Legend Snippet: ARID3B Fl induces apoptosis in ovarian carcinoma cells via TNFα/TRAIL pathways. Western blot using anti-Caspase 7, anti-Caspase 10, anti-TNF-R2, anti-TRAIL, anti-TRADD and anti- GAPDH (control) antibodies on transduced OVCA 429 ( A ) and OVCA 433 ( B ) cells. The densitometry evaluations of the western blots were analyzed (value under blot). Results were normalized to GAPDH expression and compared to parental ovarian cancer cells. Statistical analysis was performed to determine if there were any significant changes between parental ovarian cancer cells vs. GFP, ARID3B Sh or ARID3B Fl transduced ovarian cancer cells. [*p

    Techniques Used: Western Blot, Expressing

    Anti-TNFα neutralizing antibody impairs ARID3B Fl apoptotic activity. Flow cytometry analysis using Annexin V and 7-AAD was used to evaluate the apoptosis in OVCA 429 overexpressing the ARID3B Fl treated with or without 10 µg/ml anti-TNFα-neutralizing antibody. Twenty four hours pre-transduction with ARID3B Fl, OVCA 429 cells were treated with or without 10 µg/ml of human an anti-TNFα neutralizing antibody. Seventy two hours post transduction flow cytometry for Annexin V and 7-AAD was performed on OVCA 429 cells and the percentage of viable cells was expressed as the mean ± SEM of triplicate measurements. Statistical analysis was performed to determine if there were any significant changes between ARID3B Fl transduced cells vs. anti-TNFα treated ARID3B Fl transduced cells. [**p
    Figure Legend Snippet: Anti-TNFα neutralizing antibody impairs ARID3B Fl apoptotic activity. Flow cytometry analysis using Annexin V and 7-AAD was used to evaluate the apoptosis in OVCA 429 overexpressing the ARID3B Fl treated with or without 10 µg/ml anti-TNFα-neutralizing antibody. Twenty four hours pre-transduction with ARID3B Fl, OVCA 429 cells were treated with or without 10 µg/ml of human an anti-TNFα neutralizing antibody. Seventy two hours post transduction flow cytometry for Annexin V and 7-AAD was performed on OVCA 429 cells and the percentage of viable cells was expressed as the mean ± SEM of triplicate measurements. Statistical analysis was performed to determine if there were any significant changes between ARID3B Fl transduced cells vs. anti-TNFα treated ARID3B Fl transduced cells. [**p

    Techniques Used: Activity Assay, Flow Cytometry, Cytometry, Transduction

    ARID3B Fl induces apoptosis in ovarian carcinoma cells. Flow cytometry analysis using Annexin V and 7-AAD was used to evaluate the apoptosis in OVCA 429 ( A ) and OVCA 433 ( B ) overexpressing the ARID3B splice forms. OVCA 429 and OVCA 433 cells were lentivirally transduced with ARID3B Fl, ARID3B Sh and GFP (control). Flow cytometry for Annexin V and 7-AAD was performed on OVCA 429 (A) and OVCA 433 (B) the percentage of viable cells was expressed as the mean ± SEM of triplicate measurements. C . The Caspase-Glo 3/7 assay for Caspase 3/7 activities was performed on OVCA 433 and OVCA 429 ovarian cancer cells 72 h post transduction. Caspase-Glo 3/7 activity is shown in reference to parental cell and normalized to the cell count after transduction with GFP, ARID3B Sh and ARID3B Fl for 72 h. Caspase activity of parental cells was defined as 100%. Caspase activity was expressed as the mean ± SEM of triplicate measurements. D . Cell viability analysis using MTT assay was used to confirm the apoptotic phenotype of ARID3B Fl. MTT assay was performed on OVCA 429 and OVCA 433 transduced with ARID3B Fl, ARID3B Sh and GFP cells for 72 h and the percentages of viable cells (compared to parental cells) were expressed as the mean ± SEM of triplicate measurements. E . Western blot using anti-ARID3B, anti-BIM and anti- β-actin (control) antibodies on transduced OVCA 433 and OVCA 429 cells. The densitometry evaluation of the western blot analyses for BIM was analyzed (value under blot). Results were normalized to β-actin expression and compared to parental ovarian cancer cells Statistical analysis was performed to determine if there were any significant changes between parental ovarian cancer cells vs. GFP, ARID3B Sh or ARID3B Fl transduced ovarian cancer cells. [**p
    Figure Legend Snippet: ARID3B Fl induces apoptosis in ovarian carcinoma cells. Flow cytometry analysis using Annexin V and 7-AAD was used to evaluate the apoptosis in OVCA 429 ( A ) and OVCA 433 ( B ) overexpressing the ARID3B splice forms. OVCA 429 and OVCA 433 cells were lentivirally transduced with ARID3B Fl, ARID3B Sh and GFP (control). Flow cytometry for Annexin V and 7-AAD was performed on OVCA 429 (A) and OVCA 433 (B) the percentage of viable cells was expressed as the mean ± SEM of triplicate measurements. C . The Caspase-Glo 3/7 assay for Caspase 3/7 activities was performed on OVCA 433 and OVCA 429 ovarian cancer cells 72 h post transduction. Caspase-Glo 3/7 activity is shown in reference to parental cell and normalized to the cell count after transduction with GFP, ARID3B Sh and ARID3B Fl for 72 h. Caspase activity of parental cells was defined as 100%. Caspase activity was expressed as the mean ± SEM of triplicate measurements. D . Cell viability analysis using MTT assay was used to confirm the apoptotic phenotype of ARID3B Fl. MTT assay was performed on OVCA 429 and OVCA 433 transduced with ARID3B Fl, ARID3B Sh and GFP cells for 72 h and the percentages of viable cells (compared to parental cells) were expressed as the mean ± SEM of triplicate measurements. E . Western blot using anti-ARID3B, anti-BIM and anti- β-actin (control) antibodies on transduced OVCA 433 and OVCA 429 cells. The densitometry evaluation of the western blot analyses for BIM was analyzed (value under blot). Results were normalized to β-actin expression and compared to parental ovarian cancer cells Statistical analysis was performed to determine if there were any significant changes between parental ovarian cancer cells vs. GFP, ARID3B Sh or ARID3B Fl transduced ovarian cancer cells. [**p

    Techniques Used: Flow Cytometry, Cytometry, Transduction, Caspase-Glo Assay, Activity Assay, Cell Counting, MTT Assay, Western Blot, Expressing

    Expression of ARID3B splice forms in cancer cell lines. QRT-PCR for ARID3B Fl ( A ) and ARID3B Sh ( B ) was performed on total RNA isolated from the following cancer cell lines; A431 (skin), SW 480 (colon), BxPC-3 (pancreatic), PC-3 (prostate), Ca Ski (cervical), MCF-7 (breast), and three ovarian cancer cell lines, DOV13, OVCA 433 and OVCA 429. Results were normalized to 18 S rRNA expression and compared to the expression of ARID3B Fl or Sh in DOV13 ovarian cancer cells. Fold expression relative to DOV13 cells was expressed as the mean ± SEM of triplicate measurements. C . Western blot was performed for ARID3B and β-actin on cancer cell line lysates. Results were normalized to β-actin expression and compared to the expression of ARID3B Fl/Sh in DOV13 ovarian cancer cells. The densitometry evaluation of the western blot analyses for ARID3B Fl and ARID3B Sh were analyzed (value under blot). Fold expression relative to DOV13 cells was expressed as the mean of triplicate measurements.
    Figure Legend Snippet: Expression of ARID3B splice forms in cancer cell lines. QRT-PCR for ARID3B Fl ( A ) and ARID3B Sh ( B ) was performed on total RNA isolated from the following cancer cell lines; A431 (skin), SW 480 (colon), BxPC-3 (pancreatic), PC-3 (prostate), Ca Ski (cervical), MCF-7 (breast), and three ovarian cancer cell lines, DOV13, OVCA 433 and OVCA 429. Results were normalized to 18 S rRNA expression and compared to the expression of ARID3B Fl or Sh in DOV13 ovarian cancer cells. Fold expression relative to DOV13 cells was expressed as the mean ± SEM of triplicate measurements. C . Western blot was performed for ARID3B and β-actin on cancer cell line lysates. Results were normalized to β-actin expression and compared to the expression of ARID3B Fl/Sh in DOV13 ovarian cancer cells. The densitometry evaluation of the western blot analyses for ARID3B Fl and ARID3B Sh were analyzed (value under blot). Fold expression relative to DOV13 cells was expressed as the mean of triplicate measurements.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Western Blot

    EGFR regulates PEA3 binding to the ARID3B promoter. Chromatin immunoprecipitation (ChIP) analysis confirmed the binding PEA3 to the ARID3B promoter region. ChIP was performed on OVCA 429 cells serum starved (SS) for 24 h then treated with or without 20 nM EGF for 24 h. Quantitative PCR analysis was conducted for ( A ) a region of the ARID3B promoter containing ETS/PEA3 binding site and ( B ) a promoter sequence upstream of the ETS binding site. ChIP was performed using IgG (negative control) or anti-PEA3. Expression was normalized to NTC (no template control). The no template control (NTC) expression was a boundary limit (expression fold = 1), any fold-expression below this limit (expression
    Figure Legend Snippet: EGFR regulates PEA3 binding to the ARID3B promoter. Chromatin immunoprecipitation (ChIP) analysis confirmed the binding PEA3 to the ARID3B promoter region. ChIP was performed on OVCA 429 cells serum starved (SS) for 24 h then treated with or without 20 nM EGF for 24 h. Quantitative PCR analysis was conducted for ( A ) a region of the ARID3B promoter containing ETS/PEA3 binding site and ( B ) a promoter sequence upstream of the ETS binding site. ChIP was performed using IgG (negative control) or anti-PEA3. Expression was normalized to NTC (no template control). The no template control (NTC) expression was a boundary limit (expression fold = 1), any fold-expression below this limit (expression

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Sequencing, Negative Control, Expressing

    Identification of a novel alternative splice form of ARID3B (ARID3B Sh). A . Schematic representations of the proteins generated from the human ARID3B splice forms. ARID3B Sh contains a unique epitope from intron 4. B . Schematic representation of the human ARID3B splice forms pre-mRNA sequence. The unique mRNA sequence found in ARID3B Sh is depicted. C . The expression of ARID3B Sh was confirmed in ovarian cancer cell lines. The human ovarian cancer cell lines, OVCA 429 and DOV13, were serum starved (SS) for 24 h, treated with or without 20 nM EGF for an additional 24 h. RT-PCR analyses for ARID3B Sh and 18 s rRNA were performed.
    Figure Legend Snippet: Identification of a novel alternative splice form of ARID3B (ARID3B Sh). A . Schematic representations of the proteins generated from the human ARID3B splice forms. ARID3B Sh contains a unique epitope from intron 4. B . Schematic representation of the human ARID3B splice forms pre-mRNA sequence. The unique mRNA sequence found in ARID3B Sh is depicted. C . The expression of ARID3B Sh was confirmed in ovarian cancer cell lines. The human ovarian cancer cell lines, OVCA 429 and DOV13, were serum starved (SS) for 24 h, treated with or without 20 nM EGF for an additional 24 h. RT-PCR analyses for ARID3B Sh and 18 s rRNA were performed.

    Techniques Used: Generated, Sequencing, Expressing, Reverse Transcription Polymerase Chain Reaction

    Localization of ARID3B splice forms by immunofluorescence. Immunofluorescence on OVCA 433 cells expressing GFP ( a, d, g ), ARID3B Fl ( b, e, h ), and ARID3B Sh ( c, f ). Cells are untreated (0 h EGF) ( a, b, c ) or treated with EGF for 24 h ( d, e, f ). Arrow indicates plasma membrane staining. Scale bars = 50 um. Panels g and h are long exposures of panels a and b in order to better visualize membrane associated ARID3B. Scale bar = 50 uM.
    Figure Legend Snippet: Localization of ARID3B splice forms by immunofluorescence. Immunofluorescence on OVCA 433 cells expressing GFP ( a, d, g ), ARID3B Fl ( b, e, h ), and ARID3B Sh ( c, f ). Cells are untreated (0 h EGF) ( a, b, c ) or treated with EGF for 24 h ( d, e, f ). Arrow indicates plasma membrane staining. Scale bars = 50 um. Panels g and h are long exposures of panels a and b in order to better visualize membrane associated ARID3B. Scale bar = 50 uM.

    Techniques Used: Immunofluorescence, Expressing, Staining

    31) Product Images from "A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination"

    Article Title: A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx571

    PRP19 assembles with RFWD3 on RPA–ssDNA in response to DNA damage and promotes RPA ubiquitylation. ( A ) PRP19 and RFWD3 depletion perturb DNA damage-induced RPA ubiquitylation. Cells were transfected with siRNAs targeting either PRP19 or RFWD3 and a vector expressing His 6 -tagged ubiquitin, treated or not with CPT and lysed under denaturing conditions. Ni-NTA pulldown was performed to isolate ubiquitylated proteins. ( B ) Cells were transfected with SFB- (S-protein, FLAG, streptavidin-binding peptide) GFP or SFB-RFWD3 vectors and streptavidin pulldown of SFB-tagged proteins in untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( C ) Cells were transfected with SFB-GFP or SFB-PRP19 vectors and streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( D ) Cells were transfected with SFB-GFP or SFB-PRP19 and myc-RFWD3 vectors. Streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed and the indicated proteins were immunoblotted. ( E ) HeLa cells transfected with an SFB-PRP19 vector and pre-sensitized with BrdU were UV laser microirradiated. Immunofluorescence against endogenous γ-H2AX, RFWD3 and FLAG epitope was subsequently performed to monitor RFWD3 and PRP19 accrual at damage sites.
    Figure Legend Snippet: PRP19 assembles with RFWD3 on RPA–ssDNA in response to DNA damage and promotes RPA ubiquitylation. ( A ) PRP19 and RFWD3 depletion perturb DNA damage-induced RPA ubiquitylation. Cells were transfected with siRNAs targeting either PRP19 or RFWD3 and a vector expressing His 6 -tagged ubiquitin, treated or not with CPT and lysed under denaturing conditions. Ni-NTA pulldown was performed to isolate ubiquitylated proteins. ( B ) Cells were transfected with SFB- (S-protein, FLAG, streptavidin-binding peptide) GFP or SFB-RFWD3 vectors and streptavidin pulldown of SFB-tagged proteins in untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( C ) Cells were transfected with SFB-GFP or SFB-PRP19 vectors and streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed. The indicated proteins were immunoblotted. ( D ) Cells were transfected with SFB-GFP or SFB-PRP19 and myc-RFWD3 vectors. Streptavidin pulldown of SFB-tagged proteins isolated from untreated or CPT-treated cells was performed and the indicated proteins were immunoblotted. ( E ) HeLa cells transfected with an SFB-PRP19 vector and pre-sensitized with BrdU were UV laser microirradiated. Immunofluorescence against endogenous γ-H2AX, RFWD3 and FLAG epitope was subsequently performed to monitor RFWD3 and PRP19 accrual at damage sites.

    Techniques Used: Recombinase Polymerase Amplification, Transfection, Plasmid Preparation, Expressing, Cycling Probe Technology, Binding Assay, Isolation, Immunofluorescence, FLAG-tag

    32) Product Images from "An essential role of PI3K in the control of West Nile virus infection"

    Article Title: An essential role of PI3K in the control of West Nile virus infection

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03912-5

    PI3K is critical for WNV-activated nuclear translocation of IRF7. BMDMs were left untreated (mock) or infected with WNV in the presence of vehicle control (DMSO) or 5 μM of LY294002 (LY) for 24 h. ( A ) Immunoblotting analyses of IRF3, IRF7, and NF- κBp65 in the cytoplasmic (Gapdh as the housekeeping protein control) and nuclear fractions (Mcm2 as the housekeeping protein control). Blots shown are cropped from full-length. ( B ) Quantification of the nuclear immunoblot band density in ( A ) by Image J. The results are expressed as the ratio of IRF7/IRF3/p65 to Mcm2. ( C ) Immunofluorescence staining of IRF7 (red) and nuclei by DAPI (blue). Images were acquired with a Zeiss fluorescence microscope. The arrows indicate nuclear IRF7 staining. The data shown are representative of 3 independent reproducible experiments.
    Figure Legend Snippet: PI3K is critical for WNV-activated nuclear translocation of IRF7. BMDMs were left untreated (mock) or infected with WNV in the presence of vehicle control (DMSO) or 5 μM of LY294002 (LY) for 24 h. ( A ) Immunoblotting analyses of IRF3, IRF7, and NF- κBp65 in the cytoplasmic (Gapdh as the housekeeping protein control) and nuclear fractions (Mcm2 as the housekeeping protein control). Blots shown are cropped from full-length. ( B ) Quantification of the nuclear immunoblot band density in ( A ) by Image J. The results are expressed as the ratio of IRF7/IRF3/p65 to Mcm2. ( C ) Immunofluorescence staining of IRF7 (red) and nuclei by DAPI (blue). Images were acquired with a Zeiss fluorescence microscope. The arrows indicate nuclear IRF7 staining. The data shown are representative of 3 independent reproducible experiments.

    Techniques Used: Translocation Assay, Infection, Immunofluorescence, Staining, Fluorescence, Microscopy

    33) Product Images from "ZMYND8 reads the dual histone mark H3K4me1-H3K14ac to antagonize the expression of metastasis-linked genes"

    Article Title: ZMYND8 reads the dual histone mark H3K4me1-H3K14ac to antagonize the expression of metastasis-linked genes

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2016.06.035

    ZMYND8 recognizes H3K4me0-H3K14ac and H3K4me1-H3K14ac via the PHD/Bromo cassette ( A ) Schematic representation of ZMYND8-PHD/Bromo/PWWP (ZMYND8-PBP), ZMYND8-PHD/Bromo (ZMYND8-PB), and ZMYND8-PB deletion mutants. ( B ) Peptide pull-down assays using the indicated biotinylated peptides and the GST-tagged ZMYND8-PB. ( C ) Peptide pull-down assays using H3 (1-21) peptide and either the recombinant GST-ZMYND8-PB or its deletion mutants. ( D and E ) Isothermal titration calorimetry (ITC) assays to measure the interaction of the indicated peptides with ZMYND8-PB ( D ) or ZMYND8-PBP ( E ). ( F ) Peptide pull-down assays using the indicated biotinylated peptides and recombinant GST-ZMYND8-PBP. ( G .
    Figure Legend Snippet: ZMYND8 recognizes H3K4me0-H3K14ac and H3K4me1-H3K14ac via the PHD/Bromo cassette ( A ) Schematic representation of ZMYND8-PHD/Bromo/PWWP (ZMYND8-PBP), ZMYND8-PHD/Bromo (ZMYND8-PB), and ZMYND8-PB deletion mutants. ( B ) Peptide pull-down assays using the indicated biotinylated peptides and the GST-tagged ZMYND8-PB. ( C ) Peptide pull-down assays using H3 (1-21) peptide and either the recombinant GST-ZMYND8-PB or its deletion mutants. ( D and E ) Isothermal titration calorimetry (ITC) assays to measure the interaction of the indicated peptides with ZMYND8-PB ( D ) or ZMYND8-PBP ( E ). ( F ) Peptide pull-down assays using the indicated biotinylated peptides and recombinant GST-ZMYND8-PBP. ( G .

    Techniques Used: Recombinant, Isothermal Titration Calorimetry

    The chromatin landscape of ZMYND8 greatly overlaps that of JARID1D. ( A ) The genomic distribution of ChIP-seq peaks of JARID1D and ZMYND8. ChIP-Seq was performed using DU145 prostate cancer cells. ( B ) Average enrichment of JARID1D and ZMYND8 in the genic regions, including TSS and TTS. TSS, transcriptional start site; TTS, transcriptional termination site. ( C ) Heat maps of genomic co-localization of JARID1D and ZMYND8 in DU145 cells. ( D ) Venn diagram of the overlap between JARID1D and ZMYND8 peaks. ( E ) Venn diagram of genes co-occupied by JARID1D and ZMYND8. ( F ) Gene ontology analysis of genes co-occupied by JARID1D and ZMYND8.
    Figure Legend Snippet: The chromatin landscape of ZMYND8 greatly overlaps that of JARID1D. ( A ) The genomic distribution of ChIP-seq peaks of JARID1D and ZMYND8. ChIP-Seq was performed using DU145 prostate cancer cells. ( B ) Average enrichment of JARID1D and ZMYND8 in the genic regions, including TSS and TTS. TSS, transcriptional start site; TTS, transcriptional termination site. ( C ) Heat maps of genomic co-localization of JARID1D and ZMYND8 in DU145 cells. ( D ) Venn diagram of the overlap between JARID1D and ZMYND8 peaks. ( E ) Venn diagram of genes co-occupied by JARID1D and ZMYND8. ( F ) Gene ontology analysis of genes co-occupied by JARID1D and ZMYND8.

    Techniques Used: Chromatin Immunoprecipitation

    ZMYND8 knockdown increases the invasive abilities of prostate cancer cells in vitro and in vivo ( A ) Western blot and quantitative RT-PCR analysis of ZMYND8 levels following the treatment of DU145 cells with shZMYND8s (shZMYND8-95 and shZMYND8-97). ( B–D ) Effects of ZMYND8 knockdown on the proliferation ( B ), migration ( C ) and invasion ( D ) of DU145 cells. Following the cell migration and invasion assays, cells were stained with crystal violet and counted in at least five fields. ( E–G ) The effect of ZMYND8 knockdown on the in vivo metastatic abilities of DU145 cells in an intravenous mouse xenograft model. DU145 cells with stably expressing firefly luciferase were infected with lentiviruses containing scramble shRNA (shScramble) or shZMYND8-97. The representative bioluminescent images of mice (shScramble, n=6; shZMYND8, n=6) 8-10 weeks after tail vein injection are shown ( E ), and their quantified bioluminescent signals were individually plotted ( F ). Representative images of hematoxylin and eosin–stained lung tissues ( G .
    Figure Legend Snippet: ZMYND8 knockdown increases the invasive abilities of prostate cancer cells in vitro and in vivo ( A ) Western blot and quantitative RT-PCR analysis of ZMYND8 levels following the treatment of DU145 cells with shZMYND8s (shZMYND8-95 and shZMYND8-97). ( B–D ) Effects of ZMYND8 knockdown on the proliferation ( B ), migration ( C ) and invasion ( D ) of DU145 cells. Following the cell migration and invasion assays, cells were stained with crystal violet and counted in at least five fields. ( E–G ) The effect of ZMYND8 knockdown on the in vivo metastatic abilities of DU145 cells in an intravenous mouse xenograft model. DU145 cells with stably expressing firefly luciferase were infected with lentiviruses containing scramble shRNA (shScramble) or shZMYND8-97. The representative bioluminescent images of mice (shScramble, n=6; shZMYND8, n=6) 8-10 weeks after tail vein injection are shown ( E ), and their quantified bioluminescent signals were individually plotted ( F ). Representative images of hematoxylin and eosin–stained lung tissues ( G .

    Techniques Used: In Vitro, In Vivo, Western Blot, Quantitative RT-PCR, Migration, Staining, Stable Transfection, Expressing, Luciferase, Infection, shRNA, Mouse Assay, Injection

    ZMYND8 is a JARID1D-associated protein ( A ) Immunoaffinity purification and mass spectrometric analysis of JARID1D-associated proteins. JARID1D-associated proteins from nuclear extracts from FLAG-JARID1D-expressing stable H1299 cells were immunopurified with anti-FLAG (α-FLAG) affinity resins. The proteins bands were analyzed by mass spectrometry. Asterisks indicate breakdowns or non-specific polypeptides. ( B ) Schematic representation of human JARID1D, ZMYND8, and ZMYND8 deletion mutants. ( C ) Nuclear localization of JARID1D and ZMYND8 in DU145 cells. Nuclear and cytoplasmic fractions of DU145 cells were blotted with the indicated antibodies. p84 and β-actin were used as a nuclear marker and a cytoplasmic marker, respectively. WCE, whole cell extracts. ( D ) Co-immunoprecipitation between ectopically expressed FLAG-tagged JARID1D and endogenous ZMYND8 protein. Anti-FLAG-immunoprecipitated samples were blotted with anti-FLAG and anti-ZMYND8 (α-ZMYND8) antibodies. ( E ) Coimmunoprecipitation between endogenous JARID1D and ZMYND8 in DU145 cells. Anti-JARID1D (α-JARID1D)-immunoprecipitated samples were blotted with anti-JARID1D and anti-ZMYND8 antibodies. ( F ) Mapping of the ZMYND8 region responsible for the interaction with JARID1D. FLAG-JARID1D and HA-ZMYND8 (or its truncated mutants) were ectopically expressed in 293T cells. Expression levels were examined using anti-HA and anti-FLAG antibodies ( Left panel ). Following co-immunoprecipitation (IP), the eluates were examined by western blot analysis using anti-HA (α-HA) and anti-FLAG antibodies ( Right panel ). Open rectangular triangles denote specific bands, and asterisks indicate nonspecific polypeptides. ( G and H ) Mapping of the JARID1D region responsible for the interaction with ZMYND8. Recombinant full-length JARID1D and its deletion mutants were analyzed using anti-JARID1D antibody ( G ). A Co-IP assay was performed using recombinant JARID1D, JARID1D mutants, and ZMYND8 that were isolated from Sf9 or Sf21 insect cells ( H .
    Figure Legend Snippet: ZMYND8 is a JARID1D-associated protein ( A ) Immunoaffinity purification and mass spectrometric analysis of JARID1D-associated proteins. JARID1D-associated proteins from nuclear extracts from FLAG-JARID1D-expressing stable H1299 cells were immunopurified with anti-FLAG (α-FLAG) affinity resins. The proteins bands were analyzed by mass spectrometry. Asterisks indicate breakdowns or non-specific polypeptides. ( B ) Schematic representation of human JARID1D, ZMYND8, and ZMYND8 deletion mutants. ( C ) Nuclear localization of JARID1D and ZMYND8 in DU145 cells. Nuclear and cytoplasmic fractions of DU145 cells were blotted with the indicated antibodies. p84 and β-actin were used as a nuclear marker and a cytoplasmic marker, respectively. WCE, whole cell extracts. ( D ) Co-immunoprecipitation between ectopically expressed FLAG-tagged JARID1D and endogenous ZMYND8 protein. Anti-FLAG-immunoprecipitated samples were blotted with anti-FLAG and anti-ZMYND8 (α-ZMYND8) antibodies. ( E ) Coimmunoprecipitation between endogenous JARID1D and ZMYND8 in DU145 cells. Anti-JARID1D (α-JARID1D)-immunoprecipitated samples were blotted with anti-JARID1D and anti-ZMYND8 antibodies. ( F ) Mapping of the ZMYND8 region responsible for the interaction with JARID1D. FLAG-JARID1D and HA-ZMYND8 (or its truncated mutants) were ectopically expressed in 293T cells. Expression levels were examined using anti-HA and anti-FLAG antibodies ( Left panel ). Following co-immunoprecipitation (IP), the eluates were examined by western blot analysis using anti-HA (α-HA) and anti-FLAG antibodies ( Right panel ). Open rectangular triangles denote specific bands, and asterisks indicate nonspecific polypeptides. ( G and H ) Mapping of the JARID1D region responsible for the interaction with ZMYND8. Recombinant full-length JARID1D and its deletion mutants were analyzed using anti-JARID1D antibody ( G ). A Co-IP assay was performed using recombinant JARID1D, JARID1D mutants, and ZMYND8 that were isolated from Sf9 or Sf21 insect cells ( H .

    Techniques Used: Immunoaffinity Purification, Expressing, Mass Spectrometry, Marker, Immunoprecipitation, Western Blot, Recombinant, Co-Immunoprecipitation Assay, Isolation

    34) Product Images from "Amyotrophic Lateral Sclerosis associated FUS mutation shortens mitochondria and induces neurotoxicity"

    Article Title: Amyotrophic Lateral Sclerosis associated FUS mutation shortens mitochondria and induces neurotoxicity

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-33964-0

    R495X4FL partially rescues R495X induced mitochondria shortening and neurotoxicity. ( A ) Immunoblots for control, R495X- and R495X4FL-expressing neurons (left) and bar plot for normalized protein band intensities (right). Average band intensity for control was set to 1. Protein standard is shown on the right. Error bars indicate standard deviation (N = 3). P-values in one-way ANOVA are, 0.015, 0.009, 0.003, 0.731 and 0.093 for Kif5b, Dnm1l, Csde1, Mfn2 and Cox4, respectively and * p
    Figure Legend Snippet: R495X4FL partially rescues R495X induced mitochondria shortening and neurotoxicity. ( A ) Immunoblots for control, R495X- and R495X4FL-expressing neurons (left) and bar plot for normalized protein band intensities (right). Average band intensity for control was set to 1. Protein standard is shown on the right. Error bars indicate standard deviation (N = 3). P-values in one-way ANOVA are, 0.015, 0.009, 0.003, 0.731 and 0.093 for Kif5b, Dnm1l, Csde1, Mfn2 and Cox4, respectively and * p

    Techniques Used: Western Blot, Expressing, Standard Deviation

    The RNA binding profile of R495X4FL mutant. ( A ) Schematic of the NFLAG-hFUSR495X4FL (R495X4FL) mutant structure. The 4 Phenylalanines at the indicated positions of the FUS RRM were substituted to Leucines. Asterisk indicates stop codon. ( B ) Immunostaining images for anti-FLAG (left), anti-TubulinβIII (middle) and DAPI stain (right) show the cellular localization of R495X4FL in neurons. Scale bar, 10 μm. ( C ) Cell lysate (Ly) and CLIP samples (IP) with a rabbit anti-FLAG polyclonal antibody from neurons expressing R495X or R495X4FL were analyzed by immunoblot with a mouse monoclonal anti-FLAG antibody (left panel) and autoradiography (right panel). Arrow indicates FLAG tagged proteins. The numbers at the right indicate protein standards. ( D ) Immunoblot of cell lysate and IP samples using anti-FLAG antibody (top). Arrow indicates FLAG tagged proteins. The numbers at the right indicate protein standards. Asterisk indicates IgG heavy chain. RT-PCR using RNA samples from cell lysate and IP samples for the genes indicated at the right (bottom). ( E ) Gene expression levels measured by qPCR from total RNA from cell lysate. Control expression was set to 100% and the relative gene expression was measured. Error bar indicates standard deviation (N = 3). No statistically significant difference was observed for any gene. P-values in one-way ANOVA are, 0.077, 0.574, 0.250, 0.579, 0.079, 0.051 and 0.243 for Gapdh, Dnm1l, Csde1, Kif5b, Mfn2, Tubb3 and Cox4, respectively. ( F ) Gene expression levels measured by qPCR for RNA from IP samples. mRNA abundance is measured as percent of input. Lower part corresponds to [0, 4]%, while upper part to [4, 125]%. P-values in one-way ANOVA are, 0.006,
    Figure Legend Snippet: The RNA binding profile of R495X4FL mutant. ( A ) Schematic of the NFLAG-hFUSR495X4FL (R495X4FL) mutant structure. The 4 Phenylalanines at the indicated positions of the FUS RRM were substituted to Leucines. Asterisk indicates stop codon. ( B ) Immunostaining images for anti-FLAG (left), anti-TubulinβIII (middle) and DAPI stain (right) show the cellular localization of R495X4FL in neurons. Scale bar, 10 μm. ( C ) Cell lysate (Ly) and CLIP samples (IP) with a rabbit anti-FLAG polyclonal antibody from neurons expressing R495X or R495X4FL were analyzed by immunoblot with a mouse monoclonal anti-FLAG antibody (left panel) and autoradiography (right panel). Arrow indicates FLAG tagged proteins. The numbers at the right indicate protein standards. ( D ) Immunoblot of cell lysate and IP samples using anti-FLAG antibody (top). Arrow indicates FLAG tagged proteins. The numbers at the right indicate protein standards. Asterisk indicates IgG heavy chain. RT-PCR using RNA samples from cell lysate and IP samples for the genes indicated at the right (bottom). ( E ) Gene expression levels measured by qPCR from total RNA from cell lysate. Control expression was set to 100% and the relative gene expression was measured. Error bar indicates standard deviation (N = 3). No statistically significant difference was observed for any gene. P-values in one-way ANOVA are, 0.077, 0.574, 0.250, 0.579, 0.079, 0.051 and 0.243 for Gapdh, Dnm1l, Csde1, Kif5b, Mfn2, Tubb3 and Cox4, respectively. ( F ) Gene expression levels measured by qPCR for RNA from IP samples. mRNA abundance is measured as percent of input. Lower part corresponds to [0, 4]%, while upper part to [4, 125]%. P-values in one-way ANOVA are, 0.006,

    Techniques Used: RNA Binding Assay, Mutagenesis, Immunostaining, Staining, Cross-linking Immunoprecipitation, Expressing, Autoradiography, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Standard Deviation

    35) Product Images from "The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells"

    Article Title: The conserved ubiquitin-like protein Hub1 plays a critical role in splicing in human cells

    Journal: Journal of Molecular Cell Biology

    doi: 10.1093/jmcb/mju026

    Molecular mode of interaction between human Hub1 and HIND. ( A ) Mapping of the Hub1 interaction domain in hSnu66 using FLAG-immunoprecipitation of 3xFLAG-Hub1 after co-expression of GFP-tagged hSnu66 truncations or free GFP in human cells. Immunoprecipitates were immunoblotted with anti-FLAG and anti-GFP antibodies (Asterisks indicate light and heavy chains). ( B ) Crystal structure of human Hub1 (blue) in complex with HIND peptide (pink) of hSnu66 shown as a ribbon plot with a resolution of 2.0 Å. The interaction between Hub1 and the α-helical HIND peptide is mediated through a salt bridge formed by D22 of Hub1 and R127 of HIND, strengthened by hydrophobic contacts involving aliphatic fragments of residues of hSnu66's HIND (L118, I120, T123, L126, R127 (Cβ and Cγ), L130, L132, L135) and the Hub1 interface (M1, V16, L17 (Cβ, Cγ, Cδ), C18, N19 (Cβ, Cγ), L29 (Cβ, Cγ, Cδ), L30, A33). ( C ) GFP-directed immunoprecipitation of GFP fused to the HIND containing N-terminal domain (aa 1–139) of wild-type hSnu66 (WT) or Hub1-binding-deficient HIND mutant (R127A). Immunoblot detection using antibodies directed against GFP, human Hub1, or α-tubulin (control). ( D ) Co-immunoprecipitation of Hub1 with hSnu66 depends on the HIND interaction interface. GFP immunoprecipitation from U2OS cells stably expressing GFP-tagged Hub1 WT or hSnu66 binding mutant Hub1 D22A. Immunoblots were probed with anti-GFP and anti-hSnu66 antibodies with anti-U2AF65 serving as a loading control.
    Figure Legend Snippet: Molecular mode of interaction between human Hub1 and HIND. ( A ) Mapping of the Hub1 interaction domain in hSnu66 using FLAG-immunoprecipitation of 3xFLAG-Hub1 after co-expression of GFP-tagged hSnu66 truncations or free GFP in human cells. Immunoprecipitates were immunoblotted with anti-FLAG and anti-GFP antibodies (Asterisks indicate light and heavy chains). ( B ) Crystal structure of human Hub1 (blue) in complex with HIND peptide (pink) of hSnu66 shown as a ribbon plot with a resolution of 2.0 Å. The interaction between Hub1 and the α-helical HIND peptide is mediated through a salt bridge formed by D22 of Hub1 and R127 of HIND, strengthened by hydrophobic contacts involving aliphatic fragments of residues of hSnu66's HIND (L118, I120, T123, L126, R127 (Cβ and Cγ), L130, L132, L135) and the Hub1 interface (M1, V16, L17 (Cβ, Cγ, Cδ), C18, N19 (Cβ, Cγ), L29 (Cβ, Cγ, Cδ), L30, A33). ( C ) GFP-directed immunoprecipitation of GFP fused to the HIND containing N-terminal domain (aa 1–139) of wild-type hSnu66 (WT) or Hub1-binding-deficient HIND mutant (R127A). Immunoblot detection using antibodies directed against GFP, human Hub1, or α-tubulin (control). ( D ) Co-immunoprecipitation of Hub1 with hSnu66 depends on the HIND interaction interface. GFP immunoprecipitation from U2OS cells stably expressing GFP-tagged Hub1 WT or hSnu66 binding mutant Hub1 D22A. Immunoblots were probed with anti-GFP and anti-hSnu66 antibodies with anti-U2AF65 serving as a loading control.

    Techniques Used: Immunoprecipitation, Expressing, Binding Assay, Mutagenesis, Stable Transfection, Western Blot

    Hub1 is crucial for mRNA splicing of certain introns. ( A ) Alteration in alternative splicing of minigenes upon Hub1-depletion. Genomic fragments of tropomyosin 1α ( TPM , exon 3–6), myeloid cell leukemia sequence 1 ( BCL2 -related) ( Mcl-1 , exon 1–2), or fibronectin 1 ( FN1 , exon 31–34 incl. ED-A ) (see schematic exon-intron structure) expressed as minigenes in U2OS cells, and their mRNA products were analyzed by minigene-specific RT–PCR after Hub1 or control RNAi. ( B ) Detection of aberrant splicing of endogenous transcripts of v-akt murine thymoma viral oncogene homolog 1 ( AKT ), RAD23 homolog A ( RAD23A ), and Aurora kinase A ( AURKA ) after Hub1 knockdown by intron-spanning RT–PCR. ( C ) Detailed characterization of splicing specificities dependent on Hub1 and comparison to splicing factors hSnu66 and Son. Splicing of Hub1-dependent introns and flanking exons in AKT , AURKA , and MCL1 mRNAs after RNAi against Hub1, hSnu66, and Son in U2OS cells analyzed by gene-specific RT–PCR. Primer sets indicate Hub1-sensitive introns in the respective transcripts tested in RNAi experiments (red arrow heads), whereas mapping studies with PCR primers located in flanking sequences (black arrow head) detected no splicing alterations in neighboring exons/introns.
    Figure Legend Snippet: Hub1 is crucial for mRNA splicing of certain introns. ( A ) Alteration in alternative splicing of minigenes upon Hub1-depletion. Genomic fragments of tropomyosin 1α ( TPM , exon 3–6), myeloid cell leukemia sequence 1 ( BCL2 -related) ( Mcl-1 , exon 1–2), or fibronectin 1 ( FN1 , exon 31–34 incl. ED-A ) (see schematic exon-intron structure) expressed as minigenes in U2OS cells, and their mRNA products were analyzed by minigene-specific RT–PCR after Hub1 or control RNAi. ( B ) Detection of aberrant splicing of endogenous transcripts of v-akt murine thymoma viral oncogene homolog 1 ( AKT ), RAD23 homolog A ( RAD23A ), and Aurora kinase A ( AURKA ) after Hub1 knockdown by intron-spanning RT–PCR. ( C ) Detailed characterization of splicing specificities dependent on Hub1 and comparison to splicing factors hSnu66 and Son. Splicing of Hub1-dependent introns and flanking exons in AKT , AURKA , and MCL1 mRNAs after RNAi against Hub1, hSnu66, and Son in U2OS cells analyzed by gene-specific RT–PCR. Primer sets indicate Hub1-sensitive introns in the respective transcripts tested in RNAi experiments (red arrow heads), whereas mapping studies with PCR primers located in flanking sequences (black arrow head) detected no splicing alterations in neighboring exons/introns.

    Techniques Used: Sequencing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    36) Product Images from "IL-1?-specific recruitment of GCN5 histone acetyltransferase induces the release of PAF1 from chromatin for the de-repression of inflammatory response genes"

    Article Title: IL-1?-specific recruitment of GCN5 histone acetyltransferase induces the release of PAF1 from chromatin for the de-repression of inflammatory response genes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt156

    PAFc-associated PAF1 represses IL-1β–inducible genes. ( A ) Using data from Supplementary Figure S6B , the knockdown effect of individual PAFc components to IL-1β inducibility ( y -axis, relative ratio of PLAU mRNA with IL-1β to without IL-1β) and basal expression ( x -axis, relative ratio of PLAU mRNA with PAFc siRNA to control siRNA without IL-1β) are shown. ( B ) HepG2 cells were co-transfected with control or hPAF1 siRNA along with empty vector or indicated mPAFc expression vectors, and qRT–PCR for PLAU mRNA was performed. Bar indicates averages of data from three experiments; error bars represent SD. ( C ) ChIP analysis of CDC73 (as also known as parafibromin) and LEO1. HepG2 cells were stimulated with IL-1β (10 ng/ml) for 0 or 30 min. The specific protein occupancy at the target locus relative to the intergenic region was calculated and normalized to the IgG control. Bar indicates averages of data from two experiments; error bars represent standard deviation. ‘N.S.’ indicates not significant ( P > 0.05).
    Figure Legend Snippet: PAFc-associated PAF1 represses IL-1β–inducible genes. ( A ) Using data from Supplementary Figure S6B , the knockdown effect of individual PAFc components to IL-1β inducibility ( y -axis, relative ratio of PLAU mRNA with IL-1β to without IL-1β) and basal expression ( x -axis, relative ratio of PLAU mRNA with PAFc siRNA to control siRNA without IL-1β) are shown. ( B ) HepG2 cells were co-transfected with control or hPAF1 siRNA along with empty vector or indicated mPAFc expression vectors, and qRT–PCR for PLAU mRNA was performed. Bar indicates averages of data from three experiments; error bars represent SD. ( C ) ChIP analysis of CDC73 (as also known as parafibromin) and LEO1. HepG2 cells were stimulated with IL-1β (10 ng/ml) for 0 or 30 min. The specific protein occupancy at the target locus relative to the intergenic region was calculated and normalized to the IgG control. Bar indicates averages of data from two experiments; error bars represent standard deviation. ‘N.S.’ indicates not significant ( P > 0.05).

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Chromatin Immunoprecipitation, Standard Deviation

    37) Product Images from "Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression"

    Article Title: Chromatin remodeling system p300-HDAC2-Sin3A is involved in Arginine Starvation-Induced HIF-1α Degradation at the ASS1 promoter for ASS1 Derepression

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-11445-0

    Effects of ADI on the regulation of HIF-1α stability by p300, HDAC2, and Sin3A. ( a ) ChIP assay of ASS1 promoter associations of p300, HDAC2 and Sin3A in A2058 cells treated with ADI for 15 min. ( b to d ) Effects of p300, Sin3A, and HDAC2 knockdown by siRNA as indicated on the expression of other proteins in the presence and absence of ADI (0.5 μg/ml, 1 hr). ( e to g ) ChIP assays of effects of p300, Sin3A, and HDAC2 knockdown on the ASS1 promoter associations of HIF1α, PHD2, HDAC2, p300, and Sin3A as indicated Cells were transfected with given siRNA as specified for 24 hr followed by ADI treatment (0.5 μg/ml) for 15 min.
    Figure Legend Snippet: Effects of ADI on the regulation of HIF-1α stability by p300, HDAC2, and Sin3A. ( a ) ChIP assay of ASS1 promoter associations of p300, HDAC2 and Sin3A in A2058 cells treated with ADI for 15 min. ( b to d ) Effects of p300, Sin3A, and HDAC2 knockdown by siRNA as indicated on the expression of other proteins in the presence and absence of ADI (0.5 μg/ml, 1 hr). ( e to g ) ChIP assays of effects of p300, Sin3A, and HDAC2 knockdown on the ASS1 promoter associations of HIF1α, PHD2, HDAC2, p300, and Sin3A as indicated Cells were transfected with given siRNA as specified for 24 hr followed by ADI treatment (0.5 μg/ml) for 15 min.

    Techniques Used: Chromatin Immunoprecipitation, Expressing, Transfection

    Effects of histone H3 deacetylation by ADI. ( a ) A2058 cells were treated with ADI for the time intervals as indicated, or with SAHA (20 μM for 1 hr as positive control), and lamin B expression (as control for sample loading). Acetylation status of various modified histones H3 were determined. ( b ) Reduction of ASS1-promoter associations of H3K27ac and H3K14ac by ADI (0.5 μg/ml, 15 min.). ( c to e ), Effects of p300, Sin3A, and HDAC2 knockdown by siRNAs, respectively, on the expression levels of H3K14ac and H3K27ac in A2058 cells treated with or without ADI. ( f ) Western blotting analyses of H3K14ac, H3K27ac, H3K9ac, and H3K18ac expression in A2058 and ADI-resistant (ADI R variants, 58R1 to 58R3). ( g ) ChIP assay of promoter-associations of p300, HDAC2, and HIF-1α in A2058 and ADI R cells (R1 to R3). ( h ) Western blotting analyses of HIF-1α, ASS1, H3K14ac, H3K27ac, and H3 expression in 4 matched pairs of primary cell lines derived from melanoma patients before (−) and after failed (+) by ADI treatments.
    Figure Legend Snippet: Effects of histone H3 deacetylation by ADI. ( a ) A2058 cells were treated with ADI for the time intervals as indicated, or with SAHA (20 μM for 1 hr as positive control), and lamin B expression (as control for sample loading). Acetylation status of various modified histones H3 were determined. ( b ) Reduction of ASS1-promoter associations of H3K27ac and H3K14ac by ADI (0.5 μg/ml, 15 min.). ( c to e ), Effects of p300, Sin3A, and HDAC2 knockdown by siRNAs, respectively, on the expression levels of H3K14ac and H3K27ac in A2058 cells treated with or without ADI. ( f ) Western blotting analyses of H3K14ac, H3K27ac, H3K9ac, and H3K18ac expression in A2058 and ADI-resistant (ADI R variants, 58R1 to 58R3). ( g ) ChIP assay of promoter-associations of p300, HDAC2, and HIF-1α in A2058 and ADI R cells (R1 to R3). ( h ) Western blotting analyses of HIF-1α, ASS1, H3K14ac, H3K27ac, and H3 expression in 4 matched pairs of primary cell lines derived from melanoma patients before (−) and after failed (+) by ADI treatments.

    Techniques Used: Positive Control, Expressing, Modification, Western Blot, Chromatin Immunoprecipitation, Derivative Assay

    Effects of ROS on ADI-induced HIF-1α degradation. ( a ) ADI-induced HF-1α degradation is inhibited by antioxidant, NAC. A2058 cells were treated with 10 μM MG-132 in the absence or presence of ADI or NAC (1 mM) for 4-h. Expression levels of HIF-1α, hydroxylated HIF-1α, and actin were determined by Western blotting. ( b ) The antioxidant NAC suppresses ADI-induced PHD2 enzymatic activity. A2058 cells were transfected with recombinant encoding HA-PHD2. Cells were treated with NAC or ADI as indicated for 1 hr. PHD2 enzymatic activity was measured using GST-ODDD (100 ng) as a substrate and production of HO-HIF-1α (p564). ( c ) Similar to those described in ( b ) was performed using anti-oxidants Mito-TEMPO (40 μM) and TEMPO (100 μM) for 1 hr. ( d , e ) Inhibitions of HDAC2 and PHD2 interaction by TEMPO in reciprocal co-IP assays. ( f , g ), Effects of antioxidants NAC (N, 1 mM) or TEMPO (T, 100 μM)) on ADI (A)-induced ASS1 promoter association of HIF-1 α , PHD2, p300, HDAC2 and H3K27ac in A2058 cells treated with or without ADI (A, 0.5 μg/ml, 1 hr) as determined by ChIP assay.
    Figure Legend Snippet: Effects of ROS on ADI-induced HIF-1α degradation. ( a ) ADI-induced HF-1α degradation is inhibited by antioxidant, NAC. A2058 cells were treated with 10 μM MG-132 in the absence or presence of ADI or NAC (1 mM) for 4-h. Expression levels of HIF-1α, hydroxylated HIF-1α, and actin were determined by Western blotting. ( b ) The antioxidant NAC suppresses ADI-induced PHD2 enzymatic activity. A2058 cells were transfected with recombinant encoding HA-PHD2. Cells were treated with NAC or ADI as indicated for 1 hr. PHD2 enzymatic activity was measured using GST-ODDD (100 ng) as a substrate and production of HO-HIF-1α (p564). ( c ) Similar to those described in ( b ) was performed using anti-oxidants Mito-TEMPO (40 μM) and TEMPO (100 μM) for 1 hr. ( d , e ) Inhibitions of HDAC2 and PHD2 interaction by TEMPO in reciprocal co-IP assays. ( f , g ), Effects of antioxidants NAC (N, 1 mM) or TEMPO (T, 100 μM)) on ADI (A)-induced ASS1 promoter association of HIF-1 α , PHD2, p300, HDAC2 and H3K27ac in A2058 cells treated with or without ADI (A, 0.5 μg/ml, 1 hr) as determined by ChIP assay.

    Techniques Used: Expressing, Western Blot, Activity Assay, Transfection, Recombinant, Co-Immunoprecipitation Assay, Chromatin Immunoprecipitation

    38) Product Images from "Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer. Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer"

    Article Title: Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer. Novel human Ab against vascular endothelial growth factor receptor 2 shows therapeutic potential for leukemia and prostate cancer

    Journal: Cancer Science

    doi: 10.1111/cas.14208

    Selection and identification of vascular endothelial growth factor receptor‐2 (VEGFR2)‐binding single chain fragment variables (scFvs). A, After 4 rounds of biopanning for VEGFR2‐Fc recombinant protein, the phage recovery rate was increased by 3454‐fold over that of the first round. cfu, colony‐forming units. B, Comparison of the binding of selected phage clones to VEGFR2‐Fc protein by ELISA with a 1 × 10 9 cfu phage titer. CP, control phage used as a negative control; OD, optical density. C, Binding affinity of phage clones to cellular VEGFR2 was evaluated with flow cytometry of HUVECs and 1 × 10 10 cfu phage titer. D, Soluble anti‐VEGFR2 scFvs were purified and analyzed by SDS‐PAGE with Coomassie blue staining. Mr, molecular weight. E, Immunofluorescence for human tumor vasculature. Frozen sections of surgical specimens of lung cancer patients were probed with anti‐VEGFR2 scFvs, followed by anti‐E tag Ab and rhodamine‐conjugated secondary Ab. Vascular endothelium was stained with anti‐human CD31 Ab and FITC‐conjugated secondary Ab. Nuclei were stained with DAPI. Con‐scFv, control scFv. F, Competitive binding of anti‐VEGFR2 scFv and vascular endothelial growth factor‐A (VEGF‐A) was analyzed by ELISA. VEGF‐A binding to immobilized VEGFR2 in the absence of competitors was considered to be 100%. G, Phosphorylated VEGFR2 (p‐VEGFR2) expression in HUVECs treated with VEGF‐A and scFv competitors was detected by western blot. Quantification of p‐VEGFR2 was based on luminescence intensity and normalized to total VEGFR2. NHIgG, normal human IgG. Error bars, SE
    Figure Legend Snippet: Selection and identification of vascular endothelial growth factor receptor‐2 (VEGFR2)‐binding single chain fragment variables (scFvs). A, After 4 rounds of biopanning for VEGFR2‐Fc recombinant protein, the phage recovery rate was increased by 3454‐fold over that of the first round. cfu, colony‐forming units. B, Comparison of the binding of selected phage clones to VEGFR2‐Fc protein by ELISA with a 1 × 10 9 cfu phage titer. CP, control phage used as a negative control; OD, optical density. C, Binding affinity of phage clones to cellular VEGFR2 was evaluated with flow cytometry of HUVECs and 1 × 10 10 cfu phage titer. D, Soluble anti‐VEGFR2 scFvs were purified and analyzed by SDS‐PAGE with Coomassie blue staining. Mr, molecular weight. E, Immunofluorescence for human tumor vasculature. Frozen sections of surgical specimens of lung cancer patients were probed with anti‐VEGFR2 scFvs, followed by anti‐E tag Ab and rhodamine‐conjugated secondary Ab. Vascular endothelium was stained with anti‐human CD31 Ab and FITC‐conjugated secondary Ab. Nuclei were stained with DAPI. Con‐scFv, control scFv. F, Competitive binding of anti‐VEGFR2 scFv and vascular endothelial growth factor‐A (VEGF‐A) was analyzed by ELISA. VEGF‐A binding to immobilized VEGFR2 in the absence of competitors was considered to be 100%. G, Phosphorylated VEGFR2 (p‐VEGFR2) expression in HUVECs treated with VEGF‐A and scFv competitors was detected by western blot. Quantification of p‐VEGFR2 was based on luminescence intensity and normalized to total VEGFR2. NHIgG, normal human IgG. Error bars, SE

    Techniques Used: Selection, Binding Assay, Recombinant, Clone Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Flow Cytometry, Cytometry, Purification, SDS Page, Staining, Molecular Weight, Immunofluorescence, Expressing, Western Blot

    39) Product Images from "FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation"

    Article Title: FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation

    Journal: Oncotarget

    doi:

    Caspase-3 degrades FANCD2 in the absence of FANCJ and FANCJ helicase activity is not required for the stabilization of FANCD2 A. H1299 cells were transfected with control, FANCJ, and Caspase-3 siRNAs as indicated, and FANCD2 and FANCI proteins were examined by Western blots. B. The FANCJ-defective patient cell line EUFA30 was transfected with plasmids containing either wild-type or the helicase defective mutant (K52R) FANCJ. The effects of these two FANCJ variants on FANCD2 stability were determined by Western blot analysis. C. Protein extracts from H1299 cells expressing either a wild-type or helicase-defective mutant (K52R) FANCJ (both labeled with a Myc-tag) were selected to co-immunoprecipitation with antibodies directed against either FANCD2 or the Myc-tag on the 2 FANCJ variants (WT and K52R). The precipitated proteins were blotted for the corresponding partner protein (FANCD2 or Myc). FANCD2 was able to pull down both wild-type and the helicase-deficient FANCJ and FANCD2 in turn were found in immunoprecipations of both FANCJs.
    Figure Legend Snippet: Caspase-3 degrades FANCD2 in the absence of FANCJ and FANCJ helicase activity is not required for the stabilization of FANCD2 A. H1299 cells were transfected with control, FANCJ, and Caspase-3 siRNAs as indicated, and FANCD2 and FANCI proteins were examined by Western blots. B. The FANCJ-defective patient cell line EUFA30 was transfected with plasmids containing either wild-type or the helicase defective mutant (K52R) FANCJ. The effects of these two FANCJ variants on FANCD2 stability were determined by Western blot analysis. C. Protein extracts from H1299 cells expressing either a wild-type or helicase-defective mutant (K52R) FANCJ (both labeled with a Myc-tag) were selected to co-immunoprecipitation with antibodies directed against either FANCD2 or the Myc-tag on the 2 FANCJ variants (WT and K52R). The precipitated proteins were blotted for the corresponding partner protein (FANCD2 or Myc). FANCD2 was able to pull down both wild-type and the helicase-deficient FANCJ and FANCD2 in turn were found in immunoprecipations of both FANCJs.

    Techniques Used: Activity Assay, Transfection, Western Blot, Mutagenesis, Expressing, Labeling, Immunoprecipitation

    Proposed model for FANCJ stabilization of FANCD2 and FANCI FANCJ exists in a complex with both FANCD2 and FANCI in undamaged cells. This complex exists principally in the cytoplasm, but is also present in the nucleus. When there is DNA damage FANCD2 can be activated by the FA core complex and transmitted to the nucleus to participate in the formation of repair foci at the site of damage. When the K52R helicase-dead mutant, and potentially other FANCJ point mutations, is part of the complex it would protect FANCD2 but may still result in decreased DNA repair. For example, the K52R mutation has previously been shown to increase sensitivity to certain types of DNA damage, such as ionizing radiation [ 48 ], suggesting FANCJ helicase functions could be independent from stabilizing FANCD2/FANI proteins. When FANCJ is lost the unprotected FANCD2 and FANCI proteins are degraded by the ubiquitin proteasome and caspase-3.
    Figure Legend Snippet: Proposed model for FANCJ stabilization of FANCD2 and FANCI FANCJ exists in a complex with both FANCD2 and FANCI in undamaged cells. This complex exists principally in the cytoplasm, but is also present in the nucleus. When there is DNA damage FANCD2 can be activated by the FA core complex and transmitted to the nucleus to participate in the formation of repair foci at the site of damage. When the K52R helicase-dead mutant, and potentially other FANCJ point mutations, is part of the complex it would protect FANCD2 but may still result in decreased DNA repair. For example, the K52R mutation has previously been shown to increase sensitivity to certain types of DNA damage, such as ionizing radiation [ 48 ], suggesting FANCJ helicase functions could be independent from stabilizing FANCD2/FANI proteins. When FANCJ is lost the unprotected FANCD2 and FANCI proteins are degraded by the ubiquitin proteasome and caspase-3.

    Techniques Used: Mutagenesis

    FANCJ regulates FANCD2 stability, but FANCD2 has little to no effect on FANCJ protein levels A. HDF or B. H1299 cells were transfected with siRNAs for either FANCJ or FANCD2 and the levels of FANCJ, FANCD2, and FANCI were measured by Western blot. C. The average values of FANCJ, FANCD2, and FANCI in cells treated with siRNAs for FANCJ or FANCD2, from at least three independent experiments, were normalized to the levels in H1299 cells treated with control siRNA. Bars represent standard error from multiple (3–4) independent experiments. * indicates the value is significantly altered from control ( P
    Figure Legend Snippet: FANCJ regulates FANCD2 stability, but FANCD2 has little to no effect on FANCJ protein levels A. HDF or B. H1299 cells were transfected with siRNAs for either FANCJ or FANCD2 and the levels of FANCJ, FANCD2, and FANCI were measured by Western blot. C. The average values of FANCJ, FANCD2, and FANCI in cells treated with siRNAs for FANCJ or FANCD2, from at least three independent experiments, were normalized to the levels in H1299 cells treated with control siRNA. Bars represent standard error from multiple (3–4) independent experiments. * indicates the value is significantly altered from control ( P

    Techniques Used: Transfection, Western Blot

    Down-regulation or loss of FANCJ concomitantly diminishes FANCD2 and FANCI proteins in multiple cell lines HDF A. , A549 B. , H1299 C. , OV90 D. and SKVO3 E. cells were transfected with control or FANCJ siRNAs. After 48 hours whole cell lysates were collected, normalized for total protein concentration, and assessed for the levels of FANCD2, FANCI, FANCJ, and GAPDH proteins by Western blotting. F. A second, previously validated, FANCJ siRNA (FANCJ-2) was used to verify that the effects were not the result of non-specific interactions of the original siRNA.
    Figure Legend Snippet: Down-regulation or loss of FANCJ concomitantly diminishes FANCD2 and FANCI proteins in multiple cell lines HDF A. , A549 B. , H1299 C. , OV90 D. and SKVO3 E. cells were transfected with control or FANCJ siRNAs. After 48 hours whole cell lysates were collected, normalized for total protein concentration, and assessed for the levels of FANCD2, FANCI, FANCJ, and GAPDH proteins by Western blotting. F. A second, previously validated, FANCJ siRNA (FANCJ-2) was used to verify that the effects were not the result of non-specific interactions of the original siRNA.

    Techniques Used: Transfection, Protein Concentration, Western Blot

    40) Product Images from "Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers"

    Article Title: Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers

    Journal: Blood

    doi: 10.1182/blood-2005-05-2111

    GMVECs. The cells were passage 4, grown on glass coverslips. Bright field images at different magnifications show elongated cells (A; original magnification × 400) and the rounded central portion of a single cell (B; original magnification × 1000). (C-D) GMVECs were stained with rabbit anti-VWF IgG and goat anti-rabbit-IgG-Alexa 488 (green), as well as the nuclear stain, DAPI (blue). The unstimulated GMVECs contain cytoplasmic VWF-containing granules (C). The granules are secreted as long ULVWF multimeric strings (D) in response to stimulation of the GMVECs for 3 minutes with Stx-2 (1 nM). The ULVWF strings frequently become entangled, as at the top of the frame (arrow). In some Stx-1- or Stx-2-stimulated GMVECs at passage number 4 (or higher), circular conglomerates of surface VWF ( * ) that do not unfurl into strings in response to stimulation are also seen. (E) VWF multimers enriched in ULVWF are also released slowly into the culture medium in soluble form by unstimulated GMVECs over the course of 24 hours. The VWF multimers were separated by 1% agarose/SDS gel electrophoresis, and then detected by membrane transfer, a polyclonal rabbit anti-VWF IgG, goat anti-rabbit IgG-HRP and chemiluminescence. VWF released from unstimulated HUVECs over 24 hours, or present in normal human platelet-poor plasma (NP), are shown for comparison. The vertical line indicates the gel location of ULVWF forms. These photos are representative of 3 experiments.
    Figure Legend Snippet: GMVECs. The cells were passage 4, grown on glass coverslips. Bright field images at different magnifications show elongated cells (A; original magnification × 400) and the rounded central portion of a single cell (B; original magnification × 1000). (C-D) GMVECs were stained with rabbit anti-VWF IgG and goat anti-rabbit-IgG-Alexa 488 (green), as well as the nuclear stain, DAPI (blue). The unstimulated GMVECs contain cytoplasmic VWF-containing granules (C). The granules are secreted as long ULVWF multimeric strings (D) in response to stimulation of the GMVECs for 3 minutes with Stx-2 (1 nM). The ULVWF strings frequently become entangled, as at the top of the frame (arrow). In some Stx-1- or Stx-2-stimulated GMVECs at passage number 4 (or higher), circular conglomerates of surface VWF ( * ) that do not unfurl into strings in response to stimulation are also seen. (E) VWF multimers enriched in ULVWF are also released slowly into the culture medium in soluble form by unstimulated GMVECs over the course of 24 hours. The VWF multimers were separated by 1% agarose/SDS gel electrophoresis, and then detected by membrane transfer, a polyclonal rabbit anti-VWF IgG, goat anti-rabbit IgG-HRP and chemiluminescence. VWF released from unstimulated HUVECs over 24 hours, or present in normal human platelet-poor plasma (NP), are shown for comparison. The vertical line indicates the gel location of ULVWF forms. These photos are representative of 3 experiments.

    Techniques Used: Staining, SDS-Gel, Electrophoresis

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    Article Snippet: .. For immunoprecipitation of RNA–protein complexes, pre-cleared protein A-sepharose beads (SIGMA) were incubated with Gemin5 antibody (Bethyl) overnight at 4°C in a rotating wheel. .. Unbound antibody was removed, and the beads were incubated with the RNA–protein UV-crosslinking products ( ).

    Incubation:

    Article Title: Gemin5 promotes IRES interaction and translation control through its C-terminal region
    Article Snippet: .. For immunoprecipitation of RNA–protein complexes, pre-cleared protein A-sepharose beads (SIGMA) were incubated with Gemin5 antibody (Bethyl) overnight at 4°C in a rotating wheel. .. Unbound antibody was removed, and the beads were incubated with the RNA–protein UV-crosslinking products ( ).

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    Bethyl rabbit anti nfatc1 antibody
    Zp-V3 (but not Zp-P) binds <t>NFATc1.</t> (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.
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    Cif selectively inhibits USP10 activity in early endosomes. A. Cif-containing OMV reduced the activity of a 110 kDa deubiquitinating enzyme (DUB) as assessed by a DUB activity assay in cells treated with ΔCif-OMV (Control) or Cif-containing OMV (15 min treatment; see Methods and [20] , [21] , [45] ). The DUB activity assay employs a HA-UbVME probe that forms an irreversible, covalent bond only with active DUBs. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME-DUB complex using an anti-HA monoclonal antibody followed by SDS-PAGE and western blot analysis. The 110 kDa DUB was identified as USP10 by Western blot. <t>USP34</t> and USP8 were also identified in early endosomes by western blot, however, the DUB activity assay revealed that USP34 was active, but its activity was not altered by Cif. By contrast, USP8 activity was not detected. Quantitation for all western blot experiments is presented to the right. All experiments were repeated at least 3 times, * p
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    RAP80 contains a consensus SIM critical for recruitment to DSBs. (A) Diagram illustrating predicted ubiquitin-binding (blue) and SUMO-binding (orange) domains in components of the <t>BRCA1-A</t> complex. (SIM) SUMO-interacting motif; (UIM) ubiquitin-interacting
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    Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.

    Journal: PLoS Pathogens

    Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

    doi: 10.1371/journal.ppat.1007179

    Figure Lengend Snippet: Zp-V3 (but not Zp-P) binds NFATc1. (A) EMSA oligonucleotides were designed to encompass the regions of Zp-V3 and Zp-P from -155 to -127, or from -155 to -120 and radiolabeled with 32 P. The potential NFAT (in Zp-V3, not Zp-P) and AP1 binding sites (in both variants) are indicated. (B) EBV-negative BJAB cells were transfected with the Zp-V3 luciferase vector, and then treated 24 hours later with or without anti-IgM, in the presence or absence of cyclosporine A (1μM) as indicated. Luciferase activity was measured 24 hours after anti-IgM treatment and results were normalized to that of the Zp-V3 untreated condition. (set as 1). The fold increase in luciferase activity is shown for each condition (average and SD). (C) Nuclear extracts prepared from BJAB cells, treated with or without anti-IgM for 30 minutes or 6 hours, were incubated with the radiolabeled Zp-P or Zp-V3 (-155 to -127) probes in an EMSA. A protein that binds only to the Zp-V3 probe is indicated by an arrow. (D) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. Cold competitor DNA containing either the Zp-P or Zp-V3 oligonucleotides, or the consensus binding sites for the transcription factors indicated, was added in some conditions. An arrow depicts bands representing NFAT binding. (E) EMSA was performed using radiolabeled Zp-P and Zp-V3 probes (-155 to -127) and untreated nuclear BJAB extract. In some conditions, antibodies against NFATc1 or C/EBPα were added to the nuclear extract (prior to the addition of the labeled probe) as shown. An arrow depicts bands representing NFAT binding.

    Article Snippet: Supernatant was then diluted 1:5 in ChIP Dilution Buffer (16.7mM Tris pH 8.0, 167mM NaCl, 1.2mM EDTA, 1.1% Triton X-100, 0.01% SDS) and chromatin from approximately one million cells was incubated with 3ug of rabbit anti-NFATc1 antibody (Bethyl Laboratories A303-508A) or rabbit IgG control antibody (Millipore 12–370) overnight at 4°C.

    Techniques: Binding Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Incubation, Labeling

    NFATc1 and cFos cooperate to activate the Zp-V3. (A) EBV-negative BJAB cells were transfected with the wildtype Zp-V3 luciferase vector, the Zp-V3-141A, or the Zp-V3 ZIIIA mutant promoter luciferase constructs, with or without plasmids expressing cFos and/or NFATc1 as indicated. The fold increase in luciferase activity induced by co-transfection with cFos or NFATc1 vectors for each promoter construct (relative to the vector control, set as 1) is shown. Error bars indicate standard deviation. (B) Levels of transfected NFATc1 in each condition were determined by immunoblot.

    Journal: PLoS Pathogens

    Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

    doi: 10.1371/journal.ppat.1007179

    Figure Lengend Snippet: NFATc1 and cFos cooperate to activate the Zp-V3. (A) EBV-negative BJAB cells were transfected with the wildtype Zp-V3 luciferase vector, the Zp-V3-141A, or the Zp-V3 ZIIIA mutant promoter luciferase constructs, with or without plasmids expressing cFos and/or NFATc1 as indicated. The fold increase in luciferase activity induced by co-transfection with cFos or NFATc1 vectors for each promoter construct (relative to the vector control, set as 1) is shown. Error bars indicate standard deviation. (B) Levels of transfected NFATc1 in each condition were determined by immunoblot.

    Article Snippet: Supernatant was then diluted 1:5 in ChIP Dilution Buffer (16.7mM Tris pH 8.0, 167mM NaCl, 1.2mM EDTA, 1.1% Triton X-100, 0.01% SDS) and chromatin from approximately one million cells was incubated with 3ug of rabbit anti-NFATc1 antibody (Bethyl Laboratories A303-508A) or rabbit IgG control antibody (Millipore 12–370) overnight at 4°C.

    Techniques: Transfection, Luciferase, Plasmid Preparation, Mutagenesis, Construct, Expressing, Activity Assay, Cotransfection, Standard Deviation

    Converting the -141 Zp nucleotide in the intact B95.8 genome to the Zp-V3 nucleotide increases lytic protein expression in stably infected Burkitt cells. (A) EBV-negative Mutu B cells were infected with wildtype, Zp mutant, or revertant B95.8 (2089) viruses as indicated, and stably selected with hygromycin B for two months. Two different independently selected lines for each virus were then treated for two days with or without ionomycin (in the presence or absence of cyclosporine), and immunoblots were performed to detect EBNA1, EBNA2, LMP1, Z, BMRF1 (early lytic protein), p18 (late lytic protein), and actin. Kem III cell extract was included as a positive control for EBNA1, EBNA2, and LMP1. (B) Mutu cell lines containing Wt or Zp mutant viruses were nucleofected with control siRNA or NFATc1 siRNA. Ionomycin or DMSO control was added after 48 hours, and cells harvested 72 hours post-infection. Immunoblots were performed to detect NFATc1, R, BMRF1, Z, and tubulin (loading control). (C) Mutu cell lines containing Wt or Zp mutant viruses (or mock infected cells) were treated with or without anti-IgG for two days and immunoblots performed to detect BMRF1, Z, and actin (loading control). (D) Mutu cell lines containing Wt, Zp mutant, or revertant viruses were treated with or without TPA plus sodium butyrate (NaBut) (in the presence or absence of cyclosporine) for two days and immunoblots performed to detect Z expression and GAPDH (loading control). (E) ChIP assays were performed using Mutu cell lines containing Wt or Zp mutant viruses treated for three hours with ionomycin. Formaldehyde-fixed cell extracts were immunoprecipitated with control anti-IgG or NFATc1 antibody. qPCR using primers for the EBV Z promoter was performed; results shown are expressed as the amount of Zp complexed to NFATc1 ab relative to the control IgG ab. Data represent three independent experiments.

    Journal: PLoS Pathogens

    Article Title: A cancer-associated Epstein-Barr virus BZLF1 promoter variant enhances lytic infection

    doi: 10.1371/journal.ppat.1007179

    Figure Lengend Snippet: Converting the -141 Zp nucleotide in the intact B95.8 genome to the Zp-V3 nucleotide increases lytic protein expression in stably infected Burkitt cells. (A) EBV-negative Mutu B cells were infected with wildtype, Zp mutant, or revertant B95.8 (2089) viruses as indicated, and stably selected with hygromycin B for two months. Two different independently selected lines for each virus were then treated for two days with or without ionomycin (in the presence or absence of cyclosporine), and immunoblots were performed to detect EBNA1, EBNA2, LMP1, Z, BMRF1 (early lytic protein), p18 (late lytic protein), and actin. Kem III cell extract was included as a positive control for EBNA1, EBNA2, and LMP1. (B) Mutu cell lines containing Wt or Zp mutant viruses were nucleofected with control siRNA or NFATc1 siRNA. Ionomycin or DMSO control was added after 48 hours, and cells harvested 72 hours post-infection. Immunoblots were performed to detect NFATc1, R, BMRF1, Z, and tubulin (loading control). (C) Mutu cell lines containing Wt or Zp mutant viruses (or mock infected cells) were treated with or without anti-IgG for two days and immunoblots performed to detect BMRF1, Z, and actin (loading control). (D) Mutu cell lines containing Wt, Zp mutant, or revertant viruses were treated with or without TPA plus sodium butyrate (NaBut) (in the presence or absence of cyclosporine) for two days and immunoblots performed to detect Z expression and GAPDH (loading control). (E) ChIP assays were performed using Mutu cell lines containing Wt or Zp mutant viruses treated for three hours with ionomycin. Formaldehyde-fixed cell extracts were immunoprecipitated with control anti-IgG or NFATc1 antibody. qPCR using primers for the EBV Z promoter was performed; results shown are expressed as the amount of Zp complexed to NFATc1 ab relative to the control IgG ab. Data represent three independent experiments.

    Article Snippet: Supernatant was then diluted 1:5 in ChIP Dilution Buffer (16.7mM Tris pH 8.0, 167mM NaCl, 1.2mM EDTA, 1.1% Triton X-100, 0.01% SDS) and chromatin from approximately one million cells was incubated with 3ug of rabbit anti-NFATc1 antibody (Bethyl Laboratories A303-508A) or rabbit IgG control antibody (Millipore 12–370) overnight at 4°C.

    Techniques: Expressing, Stable Transfection, Infection, Mutagenesis, Western Blot, Positive Control, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

    Cif selectively inhibits USP10 activity in early endosomes. A. Cif-containing OMV reduced the activity of a 110 kDa deubiquitinating enzyme (DUB) as assessed by a DUB activity assay in cells treated with ΔCif-OMV (Control) or Cif-containing OMV (15 min treatment; see Methods and [20] , [21] , [45] ). The DUB activity assay employs a HA-UbVME probe that forms an irreversible, covalent bond only with active DUBs. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME-DUB complex using an anti-HA monoclonal antibody followed by SDS-PAGE and western blot analysis. The 110 kDa DUB was identified as USP10 by Western blot. USP34 and USP8 were also identified in early endosomes by western blot, however, the DUB activity assay revealed that USP34 was active, but its activity was not altered by Cif. By contrast, USP8 activity was not detected. Quantitation for all western blot experiments is presented to the right. All experiments were repeated at least 3 times, * p

    Journal: PLoS Pathogens

    Article Title: A Pseudomonas aeruginosa Toxin that Hijacks the Host Ubiquitin Proteolytic System

    doi: 10.1371/journal.ppat.1001325

    Figure Lengend Snippet: Cif selectively inhibits USP10 activity in early endosomes. A. Cif-containing OMV reduced the activity of a 110 kDa deubiquitinating enzyme (DUB) as assessed by a DUB activity assay in cells treated with ΔCif-OMV (Control) or Cif-containing OMV (15 min treatment; see Methods and [20] , [21] , [45] ). The DUB activity assay employs a HA-UbVME probe that forms an irreversible, covalent bond only with active DUBs. Identification of DUBs covalently linked to the HA-UbVME probe was achieved by immunoprecipitation of the HA-UbVME-DUB complex using an anti-HA monoclonal antibody followed by SDS-PAGE and western blot analysis. The 110 kDa DUB was identified as USP10 by Western blot. USP34 and USP8 were also identified in early endosomes by western blot, however, the DUB activity assay revealed that USP34 was active, but its activity was not altered by Cif. By contrast, USP8 activity was not detected. Quantitation for all western blot experiments is presented to the right. All experiments were repeated at least 3 times, * p

    Article Snippet: Antibodies and reagents The antibodies used were: mouse anti-ezrin antibody, mouse anti-G3BP1, mouse anti-GFP antibody (BD Biosciences, San Jose, CA); mouse anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-Ubiquitin antibodies (clones FK2 and FK1) (BioMol, Plymouth Meeting, PA); rabbit anti-USP10 antibody, rabbit anti-USP34, rabbit anti-USP8 (Bethyl Laboratories, Montgomery, TX); horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit secondary antibodies (Bio-Rad, Hercules, CA).

    Techniques: Activity Assay, Immunoprecipitation, SDS Page, Western Blot, Quantitation Assay

    Expression of PRMT5 and WDR77 in breast cancer ( A ). Expression analysis of 109 paired samples from The Cancer Genome Atlas (TCGA) database shows significant overexpression of PRMT5 and WDR77 in breast cancer samples relative to matched normal samples ( B ). Fold expression of WDR77 and PRMT5 in breast normal (MCF10A), ER+ (MCF7, T47D) and ER- (MDA-MB-231, HCC38) breast cancer cell lines. Normalized to GAPDH ( C ). Immunoblotting of WDR77 and PRMT5 in nuclear and cytoplasmic extracts of breast normal and cancer cell lines. Actin was used as loading control. ( D ). Fold expression (relative to GAPDH) of WDR77 and PRMT5 and ( E ). Immunoblotting of the respective proteins in sh Scrambled, sh WDR77 and sh PRMT5-treated samples. Actin was used as loading control in E ( F ). (Top panel) Annexin V staining showing increased number of apoptotic cells upon loss of WDR77 and PRMT5 . (Bottom panel) Box plots of results from apoptosis assay for three biological replicates. Student's t-test * P

    Journal: Nucleic Acids Research

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    doi: 10.1093/nar/gkx727

    Figure Lengend Snippet: Expression of PRMT5 and WDR77 in breast cancer ( A ). Expression analysis of 109 paired samples from The Cancer Genome Atlas (TCGA) database shows significant overexpression of PRMT5 and WDR77 in breast cancer samples relative to matched normal samples ( B ). Fold expression of WDR77 and PRMT5 in breast normal (MCF10A), ER+ (MCF7, T47D) and ER- (MDA-MB-231, HCC38) breast cancer cell lines. Normalized to GAPDH ( C ). Immunoblotting of WDR77 and PRMT5 in nuclear and cytoplasmic extracts of breast normal and cancer cell lines. Actin was used as loading control. ( D ). Fold expression (relative to GAPDH) of WDR77 and PRMT5 and ( E ). Immunoblotting of the respective proteins in sh Scrambled, sh WDR77 and sh PRMT5-treated samples. Actin was used as loading control in E ( F ). (Top panel) Annexin V staining showing increased number of apoptotic cells upon loss of WDR77 and PRMT5 . (Bottom panel) Box plots of results from apoptosis assay for three biological replicates. Student's t-test * P

    Article Snippet: Antibodies The following commercially available antibodies were used at the indicated concentrations for western blot: anti–β-actin (Sigma, A5441, 1:1,000), anti-WDR77 (Bethyl, A301–562A, 1:1,000), anti-PRMT5 (Bethyl, A300–850A, 1:1,000), anti-ZNF326 (Bethyl, A301–880A, 1:1,000), anti-HNRNPH1 (Bethyl, A300–511A, 1:1,000) Anti-dimethyl-Arginine, symmetric (SYM10) (Millipore, 07–412, 1:1000), Anti-Histone H3 antibody (Abcam, ab1791, 1:1000), Anti-Histone H4 (symmetric di methyl R3) (ab5823, 1:1000), Anti-Histone H2A (symmetric di methyl R3) antibody (Abcam,ab22397, 1:1000).

    Techniques: Expressing, Over Expression, Multiple Displacement Amplification, Staining, Apoptosis Assay

    Loss of PRMT5 and WDR77 leads to defects in alternative splicing and inclusion of A-T rich exons ( A and B ). (Top) Frequency of A or T upstream and downstream from splice sites of included exons (blue) excluded exons (green) and unaffected control exons (red). The dotted black line marks the meeting point of upstream and downstream datasets. (Below) Frequency of 5-base oligonucleotides in the regions around splice sites of included ( x -axis) versus control ( y -axis) exons in sh WDR77 (Left) and sh PRMT5 (Right) samples. Scatter plot of genes that are alternatively spliced and up- or downregulated upon loss of WDR77 ( C ) and PRMT5 ( D ) relative to scrambled shRNA control (Red- > 1.5-fold upregulated Blue- > 1.5-fold downregulated).

    Journal: Nucleic Acids Research

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    doi: 10.1093/nar/gkx727

    Figure Lengend Snippet: Loss of PRMT5 and WDR77 leads to defects in alternative splicing and inclusion of A-T rich exons ( A and B ). (Top) Frequency of A or T upstream and downstream from splice sites of included exons (blue) excluded exons (green) and unaffected control exons (red). The dotted black line marks the meeting point of upstream and downstream datasets. (Below) Frequency of 5-base oligonucleotides in the regions around splice sites of included ( x -axis) versus control ( y -axis) exons in sh WDR77 (Left) and sh PRMT5 (Right) samples. Scatter plot of genes that are alternatively spliced and up- or downregulated upon loss of WDR77 ( C ) and PRMT5 ( D ) relative to scrambled shRNA control (Red- > 1.5-fold upregulated Blue- > 1.5-fold downregulated).

    Article Snippet: Antibodies The following commercially available antibodies were used at the indicated concentrations for western blot: anti–β-actin (Sigma, A5441, 1:1,000), anti-WDR77 (Bethyl, A301–562A, 1:1,000), anti-PRMT5 (Bethyl, A300–850A, 1:1,000), anti-ZNF326 (Bethyl, A301–880A, 1:1,000), anti-HNRNPH1 (Bethyl, A300–511A, 1:1,000) Anti-dimethyl-Arginine, symmetric (SYM10) (Millipore, 07–412, 1:1000), Anti-Histone H3 antibody (Abcam, ab1791, 1:1000), Anti-Histone H4 (symmetric di methyl R3) (ab5823, 1:1000), Anti-Histone H2A (symmetric di methyl R3) antibody (Abcam,ab22397, 1:1000).

    Techniques: shRNA

    Alternative splicing coupled mRNA decay of transcripts upon loss of PRMT5 . qPCR analysis showing (from left to right) relative levels of exon inclusion, pre-mRNA and the mRNA/pre-mRNA ratio in sh Scrambled and sh PRMT5 samples for ( A ) REPIN1/AP4 ( B ) ST3GAL6 ( C ) PFKM ( D ) TRNAU1AP/SECP43 . The illustrations depict the gene structure with exons shown as black boxes and introns as lines. The included exons are shown in red. Arrows indicate regions to which primers were designed. Student's t -test * P

    Journal: Nucleic Acids Research

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    doi: 10.1093/nar/gkx727

    Figure Lengend Snippet: Alternative splicing coupled mRNA decay of transcripts upon loss of PRMT5 . qPCR analysis showing (from left to right) relative levels of exon inclusion, pre-mRNA and the mRNA/pre-mRNA ratio in sh Scrambled and sh PRMT5 samples for ( A ) REPIN1/AP4 ( B ) ST3GAL6 ( C ) PFKM ( D ) TRNAU1AP/SECP43 . The illustrations depict the gene structure with exons shown as black boxes and introns as lines. The included exons are shown in red. Arrows indicate regions to which primers were designed. Student's t -test * P

    Article Snippet: Antibodies The following commercially available antibodies were used at the indicated concentrations for western blot: anti–β-actin (Sigma, A5441, 1:1,000), anti-WDR77 (Bethyl, A301–562A, 1:1,000), anti-PRMT5 (Bethyl, A300–850A, 1:1,000), anti-ZNF326 (Bethyl, A301–880A, 1:1,000), anti-HNRNPH1 (Bethyl, A300–511A, 1:1,000) Anti-dimethyl-Arginine, symmetric (SYM10) (Millipore, 07–412, 1:1000), Anti-Histone H3 antibody (Abcam, ab1791, 1:1000), Anti-Histone H4 (symmetric di methyl R3) (ab5823, 1:1000), Anti-Histone H2A (symmetric di methyl R3) antibody (Abcam,ab22397, 1:1000).

    Techniques: Real-time Polymerase Chain Reaction

    ZNF326 is symmetrically dimethylated at R175 by the PRMT5/WDR77 complex ( A ). ZNF326 has two glycine-arginine rich motifs. Asterisk indicates R175 that was identified to be dimethylated ( B ). Immunoblots of immunoprecipitates showing symmetric dimethylation of ZNF326 ( C ). Representative tandem mass spectrum of the peptide GR(28.0314)GTPAYPESTFGSR {m/z:537.602 (+3). The fragment ion matching within 10 ppm are shown as either the B-ion (purple) or Y-ion (blue) series. The green dashed line indicates precursor m/z. The dotted lines in the fragmentation ladder sequence on the top the spectrum corresponds to the missing B-ion (purple) and Y-ion (blue) series ( D ). Mass Spectrometry analysis showing the relative abundance of random and dimethylated peptides in cells infected with sh Scrambled, sh WDR77 and sh PRMT5 and ( E ). Graphical representation of the same.

    Journal: Nucleic Acids Research

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    doi: 10.1093/nar/gkx727

    Figure Lengend Snippet: ZNF326 is symmetrically dimethylated at R175 by the PRMT5/WDR77 complex ( A ). ZNF326 has two glycine-arginine rich motifs. Asterisk indicates R175 that was identified to be dimethylated ( B ). Immunoblots of immunoprecipitates showing symmetric dimethylation of ZNF326 ( C ). Representative tandem mass spectrum of the peptide GR(28.0314)GTPAYPESTFGSR {m/z:537.602 (+3). The fragment ion matching within 10 ppm are shown as either the B-ion (purple) or Y-ion (blue) series. The green dashed line indicates precursor m/z. The dotted lines in the fragmentation ladder sequence on the top the spectrum corresponds to the missing B-ion (purple) and Y-ion (blue) series ( D ). Mass Spectrometry analysis showing the relative abundance of random and dimethylated peptides in cells infected with sh Scrambled, sh WDR77 and sh PRMT5 and ( E ). Graphical representation of the same.

    Article Snippet: Antibodies The following commercially available antibodies were used at the indicated concentrations for western blot: anti–β-actin (Sigma, A5441, 1:1,000), anti-WDR77 (Bethyl, A301–562A, 1:1,000), anti-PRMT5 (Bethyl, A300–850A, 1:1,000), anti-ZNF326 (Bethyl, A301–880A, 1:1,000), anti-HNRNPH1 (Bethyl, A300–511A, 1:1,000) Anti-dimethyl-Arginine, symmetric (SYM10) (Millipore, 07–412, 1:1000), Anti-Histone H3 antibody (Abcam, ab1791, 1:1000), Anti-Histone H4 (symmetric di methyl R3) (ab5823, 1:1000), Anti-Histone H2A (symmetric di methyl R3) antibody (Abcam,ab22397, 1:1000).

    Techniques: Western Blot, Sequencing, Mass Spectrometry, Infection

    Networks of top interaction partners of WDR77 in the cytoplasm and nucleus identified by LC-MS/MS ( A ). Gene ontology analysis ( B ). Protein interaction network of top 15 interacting partners of WDR77 in the cytoplasm (red lines: newly identified interactions, black lines: previously characterized interactions) ( C ). Gene ontology analysis ( D ). Protein interaction network of top 15 interacting partners of WDR77 in the nucleus (red lines: newly identified interactions, black lines: previously characterized interactions) ( E ). Immunoblots of co-immunoprecipitations of WDR77, PRMT5 and ZNF326 (IP-Immunoprecipitation, WB-western blot).

    Journal: Nucleic Acids Research

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    doi: 10.1093/nar/gkx727

    Figure Lengend Snippet: Networks of top interaction partners of WDR77 in the cytoplasm and nucleus identified by LC-MS/MS ( A ). Gene ontology analysis ( B ). Protein interaction network of top 15 interacting partners of WDR77 in the cytoplasm (red lines: newly identified interactions, black lines: previously characterized interactions) ( C ). Gene ontology analysis ( D ). Protein interaction network of top 15 interacting partners of WDR77 in the nucleus (red lines: newly identified interactions, black lines: previously characterized interactions) ( E ). Immunoblots of co-immunoprecipitations of WDR77, PRMT5 and ZNF326 (IP-Immunoprecipitation, WB-western blot).

    Article Snippet: Antibodies The following commercially available antibodies were used at the indicated concentrations for western blot: anti–β-actin (Sigma, A5441, 1:1,000), anti-WDR77 (Bethyl, A301–562A, 1:1,000), anti-PRMT5 (Bethyl, A300–850A, 1:1,000), anti-ZNF326 (Bethyl, A301–880A, 1:1,000), anti-HNRNPH1 (Bethyl, A300–511A, 1:1,000) Anti-dimethyl-Arginine, symmetric (SYM10) (Millipore, 07–412, 1:1000), Anti-Histone H3 antibody (Abcam, ab1791, 1:1000), Anti-Histone H4 (symmetric di methyl R3) (ab5823, 1:1000), Anti-Histone H2A (symmetric di methyl R3) antibody (Abcam,ab22397, 1:1000).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Western Blot, Immunoprecipitation

    The PRMT5/WDR77 complex shapes the transcriptome of MDA-MB-231 cells through methylation of ZNF326. Methylation of ZNF326 by the PRMT5/WDR77 complex is essential for Pol II transcription across A-T rich genes. Loss of PRMT5 or WDR77 leads to a loss of methylation of ZNF326 that results in slow progression of Pol II causing the inclusion of A-T rich exons in target genes. A subset of these transcripts is targeted for degradation thereby altering the shape of the transcriptome of the cell. ( A ) Represents the influence of PRMT5/WDR77 to coordinate the rate at which transcription may help determine splicing patterns, where, the absence of PRMT5/WDR77 ( B ) effects the rate and aberrant inclusion of exons.

    Journal: Nucleic Acids Research

    Article Title: The PRMT5/WDR77 complex regulates alternative splicing through ZNF326 in breast cancer

    doi: 10.1093/nar/gkx727

    Figure Lengend Snippet: The PRMT5/WDR77 complex shapes the transcriptome of MDA-MB-231 cells through methylation of ZNF326. Methylation of ZNF326 by the PRMT5/WDR77 complex is essential for Pol II transcription across A-T rich genes. Loss of PRMT5 or WDR77 leads to a loss of methylation of ZNF326 that results in slow progression of Pol II causing the inclusion of A-T rich exons in target genes. A subset of these transcripts is targeted for degradation thereby altering the shape of the transcriptome of the cell. ( A ) Represents the influence of PRMT5/WDR77 to coordinate the rate at which transcription may help determine splicing patterns, where, the absence of PRMT5/WDR77 ( B ) effects the rate and aberrant inclusion of exons.

    Article Snippet: Antibodies The following commercially available antibodies were used at the indicated concentrations for western blot: anti–β-actin (Sigma, A5441, 1:1,000), anti-WDR77 (Bethyl, A301–562A, 1:1,000), anti-PRMT5 (Bethyl, A300–850A, 1:1,000), anti-ZNF326 (Bethyl, A301–880A, 1:1,000), anti-HNRNPH1 (Bethyl, A300–511A, 1:1,000) Anti-dimethyl-Arginine, symmetric (SYM10) (Millipore, 07–412, 1:1000), Anti-Histone H3 antibody (Abcam, ab1791, 1:1000), Anti-Histone H4 (symmetric di methyl R3) (ab5823, 1:1000), Anti-Histone H2A (symmetric di methyl R3) antibody (Abcam,ab22397, 1:1000).

    Techniques: Multiple Displacement Amplification, Methylation

    RAP80 contains a consensus SIM critical for recruitment to DSBs. (A) Diagram illustrating predicted ubiquitin-binding (blue) and SUMO-binding (orange) domains in components of the BRCA1-A complex. (SIM) SUMO-interacting motif; (UIM) ubiquitin-interacting

    Journal: Science signaling

    Article Title: RNF4-Dependent Hybrid SUMO-Ubiquitin Chains are Signals for RAP80 and thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage

    doi: 10.1126/scisignal.2003485

    Figure Lengend Snippet: RAP80 contains a consensus SIM critical for recruitment to DSBs. (A) Diagram illustrating predicted ubiquitin-binding (blue) and SUMO-binding (orange) domains in components of the BRCA1-A complex. (SIM) SUMO-interacting motif; (UIM) ubiquitin-interacting

    Article Snippet: Primary antibodies were obtained from the following sources: γH2AX mouse monoclonal antibody (Millipore, Billerica, MA, clone JBW301, catalog number 05-636), BRCA1 rabbit polyclonal antibody (Bethyl Laboratories, Inc., Montgomery TX, catalog number A300-000A), RAP80 rabbit polyclonal antibody (Bethyl Laboratories, Inc., Montgomery TX, catalog number A300-763A), ubiquitin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz CA, catalog number sc-8017), and SUMO-2/3 mouse monoclonal antibody 8A2 [ ].

    Techniques: Binding Assay

    RNF4 is required for the recruitment of BRCA1 to DSBs. (A) U2OS cells were transfected with control or RNF4-specific siRNAs and subsequently treated with IR. Cells were allowed to recover for 2 h and recruitment of BRCA1 to γH2AX-labeled repair

    Journal: Science signaling

    Article Title: RNF4-Dependent Hybrid SUMO-Ubiquitin Chains are Signals for RAP80 and thereby Mediate the Recruitment of BRCA1 to Sites of DNA Damage

    doi: 10.1126/scisignal.2003485

    Figure Lengend Snippet: RNF4 is required for the recruitment of BRCA1 to DSBs. (A) U2OS cells were transfected with control or RNF4-specific siRNAs and subsequently treated with IR. Cells were allowed to recover for 2 h and recruitment of BRCA1 to γH2AX-labeled repair

    Article Snippet: Primary antibodies were obtained from the following sources: γH2AX mouse monoclonal antibody (Millipore, Billerica, MA, clone JBW301, catalog number 05-636), BRCA1 rabbit polyclonal antibody (Bethyl Laboratories, Inc., Montgomery TX, catalog number A300-000A), RAP80 rabbit polyclonal antibody (Bethyl Laboratories, Inc., Montgomery TX, catalog number A300-763A), ubiquitin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz CA, catalog number sc-8017), and SUMO-2/3 mouse monoclonal antibody 8A2 [ ].

    Techniques: Transfection, Labeling