3c reactions Search Results


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  • 94
    Thermo Fisher pentr 3c dual selection vector
    Pentr 3c Dual Selection Vector, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore hrv 3c protease site
    Hrv 3c Protease Site, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Abcam ebna 3c
    Flow cytometry analysis of β1 integrin and α4 integrin expression in control and <t>EBNA</t> 3C-expressing cell lines. Results of staining for control IgG (dotted lines), anti-β1 integrin (black fill), or anti-α4 integrin (gray
    Ebna 3c, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam sheep polyclonal anti ebna 3c antibodies
    Flow cytometry analysis of β1 integrin and α4 integrin expression in control and <t>EBNA</t> 3C-expressing cell lines. Results of staining for control IgG (dotted lines), anti-β1 integrin (black fill), or anti-α4 integrin (gray
    Sheep Polyclonal Anti Ebna 3c Antibodies, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cleavable 3c protease n terminal 6 histidine tag
    Flow cytometry analysis of β1 integrin and α4 integrin expression in control and <t>EBNA</t> 3C-expressing cell lines. Results of staining for control IgG (dotted lines), anti-β1 integrin (black fill), or anti-α4 integrin (gray
    Cleavable 3c Protease N Terminal 6 Histidine Tag, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore 3c lic vector
    This figure represents the details of the <t>3C/LIC</t> cloning strategy, PCR product containing the 5LIC extension were treated with LIC-qualified T4 DNA polymerase in the presence of dATB, and then annealed to the 3C/LIC vector.
    3c Lic Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher ecor i
    Construction of the recombinant plasmid pcDNA3.1/P12A3C. PCR ( A ), enzyme digestion ( B ). Notes: M1 and M2 indicates DNA ladder markers; Lane 1 indicates the PCR product of P12A; lane 2 indicates the PCR product of 3C; lane 3 indicates EcoR I- and Xbal I-digested pcDNA3.1/P12A3C; lane 4 indicates <t>BamH</t> I- and Xbal I-digested; lane 5 indicates BamH I- and EcoR I-digested. Abbreviation: PCR, polymerase chain reaction.
    Ecor I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1670 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega pcr clean up system
    <t>3C</t> analysis indicates contacts between the PLA2G4A and COX-2 promoters. The 3C experiments were performed using mismatch (MM)- or RNA12-treated sample (25 nM). ( A ) Diagram of COX-2/PLA2G4A loci and their intergenic region. Restriction enzyme Dpn II was used to digest chromosomal DNAs. Restriction sites for Dpn II and location of 3C primers are depicted by triangles and arrows, respectively. The 3C PCRs were performed using combinations of a constant primer (C1 or C2) and test primers (T1–8). Primer C1 and C2 are located within the constant fragment (−1077 to −354), which contains the COX-2 promoter sequence. Primers T4, T5 and T6 target the PLA2G4A promoter (∼149k bases). ( B ) Analysis of 3C <t>PCR</t> products on 2.5% agarose gels (C1 + T4, C1 + T5). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( C ) Analysis of 3C PCR products on 2.5% agarose gels (C2 + T1–8). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( D ) Quantitative analysis of relative cross-linking frequencies using PrimeTime qPCR assay. n = 4. Error bars are SEM. * P
    Pcr Clean Up System, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 22690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen quantitect sybr green pcr kit
    <t>3C</t> analysis indicates contacts between the PLA2G4A and COX-2 promoters. The 3C experiments were performed using mismatch (MM)- or RNA12-treated sample (25 nM). ( A ) Diagram of COX-2/PLA2G4A loci and their intergenic region. Restriction enzyme Dpn II was used to digest chromosomal DNAs. Restriction sites for Dpn II and location of 3C primers are depicted by triangles and arrows, respectively. The 3C PCRs were performed using combinations of a constant primer (C1 or C2) and test primers (T1–8). Primer C1 and C2 are located within the constant fragment (−1077 to −354), which contains the COX-2 promoter sequence. Primers T4, T5 and T6 target the PLA2G4A promoter (∼149k bases). ( B ) Analysis of 3C <t>PCR</t> products on 2.5% agarose gels (C1 + T4, C1 + T5). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( C ) Analysis of 3C PCR products on 2.5% agarose gels (C2 + T1–8). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( D ) Quantitative analysis of relative cross-linking frequencies using PrimeTime qPCR assay. n = 4. Error bars are SEM. * P
    Quantitect Sybr Green Pcr Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 25588 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen pcr purification minelute kit
    <t>3C</t> analysis indicates contacts between the PLA2G4A and COX-2 promoters. The 3C experiments were performed using mismatch (MM)- or RNA12-treated sample (25 nM). ( A ) Diagram of COX-2/PLA2G4A loci and their intergenic region. Restriction enzyme Dpn II was used to digest chromosomal DNAs. Restriction sites for Dpn II and location of 3C primers are depicted by triangles and arrows, respectively. The 3C PCRs were performed using combinations of a constant primer (C1 or C2) and test primers (T1–8). Primer C1 and C2 are located within the constant fragment (−1077 to −354), which contains the COX-2 promoter sequence. Primers T4, T5 and T6 target the PLA2G4A promoter (∼149k bases). ( B ) Analysis of 3C <t>PCR</t> products on 2.5% agarose gels (C1 + T4, C1 + T5). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( C ) Analysis of 3C PCR products on 2.5% agarose gels (C2 + T1–8). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( D ) Quantitative analysis of relative cross-linking frequencies using PrimeTime qPCR assay. n = 4. Error bars are SEM. * P
    Pcr Purification Minelute Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Roche gc rich pcr system
    Biochemical characterization of <t>hMex-3</t> proteins. ( A and B ) BOSC cells were transiently transfected with vectors expressing myc-tagged forms of hMex-3A, -3B and -3C. Western blot analysis was performed with anti-myc antibody. In (B) treatment of protein extracts with (+) or without (−) λ-Phosphatase. ( C ) Kinase assay. hMex-3A and -3B proteins or a control protein (Bpag1) expressed in BOSC cells were immunoprecipitated with the anti-myc antibody and incubated with kinase buffer and [γ 32 P] ATP. Labeled proteins were revealed by autoradiography. ( D ) RNA homopolymer binding assay. Proteins from indicated expression vectors were in vitro translated in the presence of [ 35 S] methionine ( top ). Binding to agarose beads coupled to poly(A) ( bottom ) RNA homopolymers is shown for in vitro translated proteins. As a negative control, a fragment of P62-sequestosome protein was incubated with RNA homopolymers in the same conditions. One-tenth of the initial translation reactions and all the bound proteins were analysed by SDS-PAGE and autoradiography. ( E ) In vivo hMex-3 binding to mRNA. BOSC cells were transiently transfected with vectors expressing myc-tagged proteins, as indicated. <t>RT-PCR</t> amplification was performed on total RNA extracted from those cells ( top left ). Western blot analysis performed with anti-myc antibody ( bottom left ). RT-PCR amplification performed on total RNA extracted from sepharose-protein A beads after immunoprecipitation by an anti-myc antibody ( right ).
    Gc Rich Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 1452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore pet 3c
    Biochemical characterization of <t>hMex-3</t> proteins. ( A and B ) BOSC cells were transiently transfected with vectors expressing myc-tagged forms of hMex-3A, -3B and -3C. Western blot analysis was performed with anti-myc antibody. In (B) treatment of protein extracts with (+) or without (−) λ-Phosphatase. ( C ) Kinase assay. hMex-3A and -3B proteins or a control protein (Bpag1) expressed in BOSC cells were immunoprecipitated with the anti-myc antibody and incubated with kinase buffer and [γ 32 P] ATP. Labeled proteins were revealed by autoradiography. ( D ) RNA homopolymer binding assay. Proteins from indicated expression vectors were in vitro translated in the presence of [ 35 S] methionine ( top ). Binding to agarose beads coupled to poly(A) ( bottom ) RNA homopolymers is shown for in vitro translated proteins. As a negative control, a fragment of P62-sequestosome protein was incubated with RNA homopolymers in the same conditions. One-tenth of the initial translation reactions and all the bound proteins were analysed by SDS-PAGE and autoradiography. ( E ) In vivo hMex-3 binding to mRNA. BOSC cells were transiently transfected with vectors expressing myc-tagged proteins, as indicated. <t>RT-PCR</t> amplification was performed on total RNA extracted from those cells ( top left ). Western blot analysis performed with anti-myc antibody ( bottom left ). RT-PCR amplification performed on total RNA extracted from sepharose-protein A beads after immunoprecipitation by an anti-myc antibody ( right ).
    Pet 3c, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Qiagen real time qrt pcr 3c
    Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control <t>PCR</t> panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 <t>qRT-PCR</t> at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).
    Real Time Qrt Pcr 3c, supplied by Qiagen, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 3c dna
    Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control <t>PCR</t> panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 <t>qRT-PCR</t> at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).
    3c Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology sema3c
    R1881-induction of <t>SEMA3C</t> expression is GATA2-dependent We examined R1881-induced expression of SEMA3C in the absence of GATA2 to confirm findings from previous microarray studies showing that knockdown of GATA2 decreases SEMA3C expression. When compared to LNCaP treated with scrambled siRNA (siSCX), knockdown of GATA2 (siGATA2) triggered a significant decrease in basal SEMA3C expression and completely attenuated R1881-mediated dose-dependent induction of SEMA3C as shown by qPCR A . These observations were confirmed at the protein level by Western blot analysis B . of both conditioned media (CM) and whole cell extract (WCE) where total actin served as loading control. In chromatin immunoprecipitation assays, SEMA3C ARE amplicon was shown be enriched in GATA2 immunoprecipitates of lysates from LNCaP cells treated with R1881 as shown by end-point C . and qPCR D . indicating an R1881-dependent recruitment of GATA2 to the SEMA3C intron 2 ARE. Input = 1% input, Isotype = isotype-matched control antibody, IP: GATA2 = GATA2 immunoprecipitates. PCR primers for these experiments were the same as those for Figure 3 and map to the SEMA3C intron 2 ARE. ChIP qPCR values represent a fold enrichment over isotype control of the same treatment condition. Chromatin immunoprecipitation assays previously showing R1881-induced recruitment of AR to the SEMA3C ARE were repeated in the presence of siRNA to GATA2 E . Values represent a fold enrichment over isotype control of the same treatment condition. Data represent mean, ± SD; * p
    Sema3c, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 123 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Illumina Inc pe 2 0 primer
    R1881-induction of <t>SEMA3C</t> expression is GATA2-dependent We examined R1881-induced expression of SEMA3C in the absence of GATA2 to confirm findings from previous microarray studies showing that knockdown of GATA2 decreases SEMA3C expression. When compared to LNCaP treated with scrambled siRNA (siSCX), knockdown of GATA2 (siGATA2) triggered a significant decrease in basal SEMA3C expression and completely attenuated R1881-mediated dose-dependent induction of SEMA3C as shown by qPCR A . These observations were confirmed at the protein level by Western blot analysis B . of both conditioned media (CM) and whole cell extract (WCE) where total actin served as loading control. In chromatin immunoprecipitation assays, SEMA3C ARE amplicon was shown be enriched in GATA2 immunoprecipitates of lysates from LNCaP cells treated with R1881 as shown by end-point C . and qPCR D . indicating an R1881-dependent recruitment of GATA2 to the SEMA3C intron 2 ARE. Input = 1% input, Isotype = isotype-matched control antibody, IP: GATA2 = GATA2 immunoprecipitates. PCR primers for these experiments were the same as those for Figure 3 and map to the SEMA3C intron 2 ARE. ChIP qPCR values represent a fold enrichment over isotype control of the same treatment condition. Chromatin immunoprecipitation assays previously showing R1881-induced recruitment of AR to the SEMA3C ARE were repeated in the presence of siRNA to GATA2 E . Values represent a fold enrichment over isotype control of the same treatment condition. Data represent mean, ± SD; * p
    Pe 2 0 Primer, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Flow cytometry analysis of β1 integrin and α4 integrin expression in control and EBNA 3C-expressing cell lines. Results of staining for control IgG (dotted lines), anti-β1 integrin (black fill), or anti-α4 integrin (gray

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: Flow cytometry analysis of β1 integrin and α4 integrin expression in control and EBNA 3C-expressing cell lines. Results of staining for control IgG (dotted lines), anti-β1 integrin (black fill), or anti-α4 integrin (gray

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques: Flow Cytometry, Cytometry, Expressing, Staining

    EBNA 3C associates with ITGA4 , ITGB1 , ADAM28 , and ADAMDEC1 regulatory regions.

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C associates with ITGA4 , ITGB1 , ADAM28 , and ADAMDEC1 regulatory regions.

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques:

    EBNA 3C binding and histone modification at ITGA4 . (A) EBNA 3 binding at the ITGA4 locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP using anti-H3K27me3

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C binding and histone modification at ITGA4 . (A) EBNA 3 binding at the ITGA4 locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP using anti-H3K27me3

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques: Binding Assay, Modification, Chromatin Immunoprecipitation, Expressing, Stable Transfection

    CXCL10 and CXCL11 are downregulated by EBNA 3C. Real-time PCR analysis of CXCL10 (A and B) and CXCL11 (C and D) mRNA expression in EBV nuclear antigen-expressing cells. Results are shown as means ± standard deviations of three independent experiments

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: CXCL10 and CXCL11 are downregulated by EBNA 3C. Real-time PCR analysis of CXCL10 (A and B) and CXCL11 (C and D) mRNA expression in EBV nuclear antigen-expressing cells. Results are shown as means ± standard deviations of three independent experiments

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Histone modification at the CXCL gene cluster in EBNA 3C-expressing cells. (A and B) ChIP using anti-histone H3K27me3 antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines analyzed with primers across the

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: Histone modification at the CXCL gene cluster in EBNA 3C-expressing cells. (A and B) ChIP using anti-histone H3K27me3 antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines analyzed with primers across the

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques: Modification, Expressing, Chromatin Immunoprecipitation, Stable Transfection

    EBNA 3C expression reduces chemotaxis of CXCR3-expressing cells. Chemotaxis assays using cell culture supernatant from control or EBNA 3C-expressing cells were performed. Shown is the migration of CXCR3-transfected cells expressed relative to the number

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C expression reduces chemotaxis of CXCR3-expressing cells. Chemotaxis assays using cell culture supernatant from control or EBNA 3C-expressing cells were performed. Shown is the migration of CXCR3-transfected cells expressed relative to the number

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques: Expressing, Chemotaxis Assay, Cell Culture, Migration, Transfection

    EBNA 3C binding and histone modification at ITGB1 . (A) EBNA 3 binding at the ITGB1 locus. The numbers of sequencing reads from EBNA 3-enriched DNA are plotted per million background-subtracted total reads and aligned with the human genome. The positions

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C binding and histone modification at ITGB1 . (A) EBNA 3 binding at the ITGB1 locus. The numbers of sequencing reads from EBNA 3-enriched DNA are plotted per million background-subtracted total reads and aligned with the human genome. The positions

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques: Binding Assay, Modification, Sequencing

    EBNA 3C binding and histone modification at the ADAM gene cluster. (A) EBNA 3 binding at the ADAM gene locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C binding and histone modification at the ADAM gene cluster. (A) EBNA 3 binding at the ADAM gene locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP

    Article Snippet: An input control sample was removed, and EBNA 3C was then precipitated by overnight rotation at 4°C with 60 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam).

    Techniques: Binding Assay, Modification, Chromatin Immunoprecipitation, Expressing, Stable Transfection

    Validation of established EBNA3C HDmut and EBNA3C W227S LCLs. (A) Cell proliferation assay at day 36 after infection of primary B cells with the wild-type (WT), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), or EBNA3C RBPJ BM (3C RBPJ BM) recombinant virus. Live cells were analyzed for proliferation by measuring EdU incorporation and DNA content (by use of FxCycle Far Red DNA stain). Gates show the populations of cells in the sub-G 1 , G 1 , S, and G 2 /M phases. Data are representative of three independent infections. (B) Expression of Epstein-Barr virus latency-associated proteins EBNA3A, EBNA3B, EBNA3C, EBNA1, EBNA2, LMP1, and EBNA-LP, as well as RBPJ and γ-tubulin, was demonstrated by Western blotting of extracts of LCLs established from primary B cell infections with the wild-type (WT), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), and EBNA3C RBPJ BM (3C RBPJ BM) EBVs used for panel A. (C) Immunoprecipitation (IP) of RBPJ or an antibody isotype control (IgG) in the WT, 3C HDmut, 3C W227S, and 3C RBPJ BM LCLs and Western blotting of EBNA3C, as indicated. Input represents 10% of the lysate used in IPs. Pulldown of each EBNA3C mutant was quantified by use of ImageJ software, and nonspecific pulldown (IgG background) was subtracted. Each IP was normalized to its input and expressed as the percent relative IP compared to the positive-control level (EBNA3C WT).

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: Validation of established EBNA3C HDmut and EBNA3C W227S LCLs. (A) Cell proliferation assay at day 36 after infection of primary B cells with the wild-type (WT), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), or EBNA3C RBPJ BM (3C RBPJ BM) recombinant virus. Live cells were analyzed for proliferation by measuring EdU incorporation and DNA content (by use of FxCycle Far Red DNA stain). Gates show the populations of cells in the sub-G 1 , G 1 , S, and G 2 /M phases. Data are representative of three independent infections. (B) Expression of Epstein-Barr virus latency-associated proteins EBNA3A, EBNA3B, EBNA3C, EBNA1, EBNA2, LMP1, and EBNA-LP, as well as RBPJ and γ-tubulin, was demonstrated by Western blotting of extracts of LCLs established from primary B cell infections with the wild-type (WT), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), and EBNA3C RBPJ BM (3C RBPJ BM) EBVs used for panel A. (C) Immunoprecipitation (IP) of RBPJ or an antibody isotype control (IgG) in the WT, 3C HDmut, 3C W227S, and 3C RBPJ BM LCLs and Western blotting of EBNA3C, as indicated. Input represents 10% of the lysate used in IPs. Pulldown of each EBNA3C mutant was quantified by use of ImageJ software, and nonspecific pulldown (IgG background) was subtracted. Each IP was normalized to its input and expressed as the percent relative IP compared to the positive-control level (EBNA3C WT).

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Proliferation Assay, Infection, Recombinant, Staining, Expressing, Western Blot, Immunoprecipitation, Mutagenesis, Software, Positive Control

    The TFGC motif of EBNA3C is important for its transcriptional repression activity. CD19 + purified B cells were infected with either the wild-type (WT), EBNA3C knockout (3C KO), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), or EBNA3C RBPJ BM (3C RBPJ BM) recombinant EBV and cultured for 30 days. RNA samples were taken at the indicated times after infection, and qPCR analysis performed on each. Gene expression of COBLL1 (A), ADAM28 (B), ADAMDEC1 (C), and ALAS1 (D) was normalized to that of the endogenous control GNB2L1 and is shown relative to that in uninfected primary B cells. Data are representative of three independent time course experiments.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: The TFGC motif of EBNA3C is important for its transcriptional repression activity. CD19 + purified B cells were infected with either the wild-type (WT), EBNA3C knockout (3C KO), EBNA3C revertant (3C Rev), EBNA3C HDmut (3C HDmut), EBNA3C W227S (3C W227S), or EBNA3C RBPJ BM (3C RBPJ BM) recombinant EBV and cultured for 30 days. RNA samples were taken at the indicated times after infection, and qPCR analysis performed on each. Gene expression of COBLL1 (A), ADAM28 (B), ADAMDEC1 (C), and ALAS1 (D) was normalized to that of the endogenous control GNB2L1 and is shown relative to that in uninfected primary B cells. Data are representative of three independent time course experiments.

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Activity Assay, Purification, Infection, Knock-Out, Recombinant, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    The EBNA3C protein interacts with the histone demethylase KDM2B. (A) KDM2B and γ-tubulin protein expression in LCLs WT, EBNA3C HDmut, and EBNA3C W227S. (B) Immunoprecipitation (IP) of KDM2B or an antibody isotype control (IgG) in the LCL EBNA3C WT used for panel A and Western blotting of EBNA3C and KDM2B, as indicated. Input represents 10% of the lysate used for IP. (C) Immunoprecipitation of KDM2B or an antibody isotype control (IgG) in the LCLs EBNA3C HDmut and EBNA3C W227S used for panel A and Western blotting of EBNA3C, as indicated. Input represents 10% of the lysate used in IPs. Pulldown of each EBNA3C mutant was quantified by use of ImageJ software, and nonspecific pulldown (IgG background) was subtracted. Each IP was normalized to its input and expressed as the percent relative IP compared to the positive-control level (EBNA3C WT; see panel B).

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: The EBNA3C protein interacts with the histone demethylase KDM2B. (A) KDM2B and γ-tubulin protein expression in LCLs WT, EBNA3C HDmut, and EBNA3C W227S. (B) Immunoprecipitation (IP) of KDM2B or an antibody isotype control (IgG) in the LCL EBNA3C WT used for panel A and Western blotting of EBNA3C and KDM2B, as indicated. Input represents 10% of the lysate used for IP. (C) Immunoprecipitation of KDM2B or an antibody isotype control (IgG) in the LCLs EBNA3C HDmut and EBNA3C W227S used for panel A and Western blotting of EBNA3C, as indicated. Input represents 10% of the lysate used in IPs. Pulldown of each EBNA3C mutant was quantified by use of ImageJ software, and nonspecific pulldown (IgG background) was subtracted. Each IP was normalized to its input and expressed as the percent relative IP compared to the positive-control level (EBNA3C WT; see panel B).

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Software, Positive Control

    Epigenetic changes at sites within the COBLL1 and ADAM28-ADAMDEC1 loci in EBNA3C mutant LCLs. (A) ChIP for H3K27me3 on the WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM LCLs at locations across the COBLL1 locus, at the Myogenin promoter (MYOG), or at the GAPDH promoter, as indicated. As a control, a similar ChIP was performed using the conditional LCL 3CHT Never HT. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. (B) Same as panel A, but using primers across the ADAM28-ADAMDEC1 locus. (C and D) Same as panels A and B, respectively, but using an anti-H3K4me3 antibody for ChIP analyses. (E and F) Same as panels A and B, respectively, but using an anti-H3K27Ac antibody for ChIP analyses.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: Epigenetic changes at sites within the COBLL1 and ADAM28-ADAMDEC1 loci in EBNA3C mutant LCLs. (A) ChIP for H3K27me3 on the WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM LCLs at locations across the COBLL1 locus, at the Myogenin promoter (MYOG), or at the GAPDH promoter, as indicated. As a control, a similar ChIP was performed using the conditional LCL 3CHT Never HT. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. (B) Same as panel A, but using primers across the ADAM28-ADAMDEC1 locus. (C and D) Same as panels A and B, respectively, but using an anti-H3K4me3 antibody for ChIP analyses. (E and F) Same as panels A and B, respectively, but using an anti-H3K27Ac antibody for ChIP analyses.

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Mutagenesis, Chromatin Immunoprecipitation

    EBNA3C HDmut efficiently binds to RBPJ and recruits it to target genes. (A) ChIP-qPCR analyses using anti-EBNA3C to precipitate EBNA3C protein and chromatin associated with it from WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM LCLs. As a control for antibody specificity, a similar ChIP was performed using the conditional LCL EBNA3C-HT never cultured with HT (LCL 3CHT Never HT). Primers for the Myogenin promoter (MYOG) as well as for a region inside the COBLL1 genomic locus ( COBLL1 control) were used as negative controls for qPCR, whereas primers for the EBNA3C binding site at COBLL1 ( COBLL1 peak) were used as positive controls for EBNA3C WT binding. ChIP values represent mean enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. (B) Same as panel A, but using control primers for the ADAM cluster region ( ADAM control) as well as primers for the ADAM28-ADAMDEC1 intergenic enhancer ( ADAM peak). (C) Same as panel A, but using anti-RBPJ antibody for ChIP analyses. (D) Same as panel B, but using anti-RBPJ antibody for ChIP analyses.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: EBNA3C HDmut efficiently binds to RBPJ and recruits it to target genes. (A) ChIP-qPCR analyses using anti-EBNA3C to precipitate EBNA3C protein and chromatin associated with it from WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM LCLs. As a control for antibody specificity, a similar ChIP was performed using the conditional LCL EBNA3C-HT never cultured with HT (LCL 3CHT Never HT). Primers for the Myogenin promoter (MYOG) as well as for a region inside the COBLL1 genomic locus ( COBLL1 control) were used as negative controls for qPCR, whereas primers for the EBNA3C binding site at COBLL1 ( COBLL1 peak) were used as positive controls for EBNA3C WT binding. ChIP values represent mean enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. (B) Same as panel A, but using control primers for the ADAM cluster region ( ADAM control) as well as primers for the ADAM28-ADAMDEC1 intergenic enhancer ( ADAM peak). (C) Same as panel A, but using anti-RBPJ antibody for ChIP analyses. (D) Same as panel B, but using anti-RBPJ antibody for ChIP analyses.

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Cell Culture, Binding Assay

    Generation of RBPJ interaction mutants of EBNA3C recombinant EBV BACs. (A) Schematic representation of mutations of the two RBPJ interaction motifs found in EBNA3C. The 209 TFGC 212 and 226 VWTP 229 motifs of EBNA3C were mutated to 209 AAAA 212 and W227S, respectively. Mutations were introduced using an In-Fusion-based mutagenesis process, with the 209 AAAA 212 mutation (homology domain [HD] mutant) introducing a NotI restriction site, whereas the W227S mutation introduced a SalI restriction site. The nucleotide sequence of wild-type (WT) EBNA3C is shown in red, whereas the HDmut and W227S sequences are shown in orange and blue, respectively. Both RBPJ interaction mutants of EBNA3C were introduced into the B95.8 EBV BAC by RecA-mediated homologous recombination. (B) BACs of WT and newly created RBPJ interaction mutant EBNA3C (HDmut and W227S) EBVs were analyzed by restriction digestion and pulsed-field gel electrophoresis. NotI restriction digestion showed that introduction of the 209 AAAA 212 mutation created an additional NotI restriction site, cutting the 36,807-bp WT band into two bands, of 18,540 bp and 18,267 bp. EcoRI and AgeI restriction enzyme digestion revealed that the integrity of the EBNA3C mutant EBV BACs was maintained during the recombination process compared to that of the WT EBV BAC. SalI restriction digestion showed that introduction of the W227S mutation created an additional SalI restriction site, cutting the wild-type, 29,695-bp band into a 23,521-bp and a 6,174-bp band.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: Generation of RBPJ interaction mutants of EBNA3C recombinant EBV BACs. (A) Schematic representation of mutations of the two RBPJ interaction motifs found in EBNA3C. The 209 TFGC 212 and 226 VWTP 229 motifs of EBNA3C were mutated to 209 AAAA 212 and W227S, respectively. Mutations were introduced using an In-Fusion-based mutagenesis process, with the 209 AAAA 212 mutation (homology domain [HD] mutant) introducing a NotI restriction site, whereas the W227S mutation introduced a SalI restriction site. The nucleotide sequence of wild-type (WT) EBNA3C is shown in red, whereas the HDmut and W227S sequences are shown in orange and blue, respectively. Both RBPJ interaction mutants of EBNA3C were introduced into the B95.8 EBV BAC by RecA-mediated homologous recombination. (B) BACs of WT and newly created RBPJ interaction mutant EBNA3C (HDmut and W227S) EBVs were analyzed by restriction digestion and pulsed-field gel electrophoresis. NotI restriction digestion showed that introduction of the 209 AAAA 212 mutation created an additional NotI restriction site, cutting the 36,807-bp WT band into two bands, of 18,540 bp and 18,267 bp. EcoRI and AgeI restriction enzyme digestion revealed that the integrity of the EBNA3C mutant EBV BACs was maintained during the recombination process compared to that of the WT EBV BAC. SalI restriction digestion showed that introduction of the W227S mutation created an additional SalI restriction site, cutting the wild-type, 29,695-bp band into a 23,521-bp and a 6,174-bp band.

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Recombinant, Mutagenesis, Sequencing, BAC Assay, Homologous Recombination, Pulsed-Field Gel, Electrophoresis

    COBLL1 , ADAM28 , and ADAMDEC1 regulation in EBNA3C lentiviral expression system in LCLs. (A) EBNA3C, mCherry, and γ-tubulin protein expression in the LCL 3CHT Never HT infected with lentiviruses expressing either mCherry, EBNA3C WT, EBNA3C HDmut, or EBNA3C W227S for 30 days. A protein extract of LCL WT was used as a positive control for EBNA3C expression. The residual expression of EBNA3C-HT is shown by a black arrow. (B to E) Expression levels of COBLL1 (B), ADAM28 (C), ADAMDEC1 (D), and ALAS1 (E) were determined for the LCLs used for panel A. (F) The H3K4me3 level was assessed by ChIP assay of LCLs infected with the lentiviruses used for panel A. Primer pairs across the COBLL1 and ADAM28-ADAMDEC1 loci were used, as well as primers for Myogenin (MYOG) and GAPDH , as negative and positive controls, respectively. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. Data are representative of one of at least two independent experiments.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: COBLL1 , ADAM28 , and ADAMDEC1 regulation in EBNA3C lentiviral expression system in LCLs. (A) EBNA3C, mCherry, and γ-tubulin protein expression in the LCL 3CHT Never HT infected with lentiviruses expressing either mCherry, EBNA3C WT, EBNA3C HDmut, or EBNA3C W227S for 30 days. A protein extract of LCL WT was used as a positive control for EBNA3C expression. The residual expression of EBNA3C-HT is shown by a black arrow. (B to E) Expression levels of COBLL1 (B), ADAM28 (C), ADAMDEC1 (D), and ALAS1 (E) were determined for the LCLs used for panel A. (F) The H3K4me3 level was assessed by ChIP assay of LCLs infected with the lentiviruses used for panel A. Primer pairs across the COBLL1 and ADAM28-ADAMDEC1 loci were used, as well as primers for Myogenin (MYOG) and GAPDH , as negative and positive controls, respectively. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. Data are representative of one of at least two independent experiments.

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Expressing, Infection, Positive Control, Chromatin Immunoprecipitation

    EBNA3C HDmut still recruits Polycomb proteins to target genes. (A and B) BMI1 and SUZ12 were immunoprecipitated (IP) from a WT LCL protein extract by use of anti-BMI1 (A), anti-SUZ12 (B), or an antibody isotype control (IgG). Immunoprecipitated and coimmunoprecipitated proteins were analyzed by Western blotting. Input corresponds to 10% of the cell extract used for IPs. (C) BMI1 and γ-tubulin protein expression in LCLs WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM. (D) Immunoprecipitation performed with anti-BMI1 or an antibody isotype control (IgG) on extracts of the 3C HDmut, 3C W227S, and 3C RBPJ BM LCLs and Western blotting of EBNA3C, as indicated. Input represents 10% of the lysate used in IPs. Pulldown of each EBNA3C mutant was quantified by use of ImageJ software, and nonspecific pulldown (IgG background) was subtracted. Each IP was normalized to its input and expressed as the percent relative IP compared to the positive-control level (EBNA3C WT; see panel A). (E and F) ChIP was performed for BMI1 (E) and SUZ12 (F) within the COBLL1 locus on extracts of the WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM LCLs as well as the LCL EBNA3C-HT Never HT. A primer pair for the Myogenin promoter (MYOG) was used as a negative control. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. (G and H) Same as panels E and F, but using primers across the ADAM28-ADAMDEC1 locus.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: EBNA3C HDmut still recruits Polycomb proteins to target genes. (A and B) BMI1 and SUZ12 were immunoprecipitated (IP) from a WT LCL protein extract by use of anti-BMI1 (A), anti-SUZ12 (B), or an antibody isotype control (IgG). Immunoprecipitated and coimmunoprecipitated proteins were analyzed by Western blotting. Input corresponds to 10% of the cell extract used for IPs. (C) BMI1 and γ-tubulin protein expression in LCLs WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM. (D) Immunoprecipitation performed with anti-BMI1 or an antibody isotype control (IgG) on extracts of the 3C HDmut, 3C W227S, and 3C RBPJ BM LCLs and Western blotting of EBNA3C, as indicated. Input represents 10% of the lysate used in IPs. Pulldown of each EBNA3C mutant was quantified by use of ImageJ software, and nonspecific pulldown (IgG background) was subtracted. Each IP was normalized to its input and expressed as the percent relative IP compared to the positive-control level (EBNA3C WT; see panel A). (E and F) ChIP was performed for BMI1 (E) and SUZ12 (F) within the COBLL1 locus on extracts of the WT, EBNA3C HDmut, EBNA3C W227S, and EBNA3C RBPJ BM LCLs as well as the LCL EBNA3C-HT Never HT. A primer pair for the Myogenin promoter (MYOG) was used as a negative control. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. (G and H) Same as panels E and F, but using primers across the ADAM28-ADAMDEC1 locus.

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

    Techniques: Immunoprecipitation, Western Blot, Expressing, Mutagenesis, Software, Positive Control, Chromatin Immunoprecipitation, Negative Control

    KDM2B is required for efficient repression of COBLL1 , ADAM28 , and ADAMDEC1 by EBNA3C. (A) Schematic of time course experiment. LCL 3CHT Never HT cells were infected with lentiviruses carrying either a control nontargeting (NT) shRNA or shRNAs directed against KDM2B. After 2 days, puromycin was added to the culture medium to select the infected cells. HT was then added to half of the culture (+HT), and the other half was left without HT (Never HT). After 5 days of HT treatment, cells were harvested. (B) Western blots of extracts from the time course experiment described for panel A, showing efficient knockdown of KDM2B after infection with lentiviruses carrying two different shRNAs against KDM2B (shKDM2B-1 and shKDM2B-2) and showing stabilization of EBNA3C after addition of HT. (C to F) Expression levels of COBLL1 (C), ADAM28 (D), ADAMDEC1 (E), and ALAS1 (F) were measured by qPCR with the cells used for panel B. Gene expression was normalized to that of the endogenous control GNB2L1 and is shown relative to that of each cell line never treated with HT (Never HT). (G) The H3K4me3 level was assessed by ChIP assay of the LCLs 3CHT shNT, 3CHT shKDM2B-1, and 3CHT shKDM2B-2 used for panel B. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. Data are representative of one of two independent infections.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Nuclear Antigen 3C Inhibits Expression of COBLL1 and the ADAM28-ADAMDEC1 Locus via Interaction with the Histone Lysine Demethylase KDM2B

    doi: 10.1128/JVI.01362-18

    Figure Lengend Snippet: KDM2B is required for efficient repression of COBLL1 , ADAM28 , and ADAMDEC1 by EBNA3C. (A) Schematic of time course experiment. LCL 3CHT Never HT cells were infected with lentiviruses carrying either a control nontargeting (NT) shRNA or shRNAs directed against KDM2B. After 2 days, puromycin was added to the culture medium to select the infected cells. HT was then added to half of the culture (+HT), and the other half was left without HT (Never HT). After 5 days of HT treatment, cells were harvested. (B) Western blots of extracts from the time course experiment described for panel A, showing efficient knockdown of KDM2B after infection with lentiviruses carrying two different shRNAs against KDM2B (shKDM2B-1 and shKDM2B-2) and showing stabilization of EBNA3C after addition of HT. (C to F) Expression levels of COBLL1 (C), ADAM28 (D), ADAMDEC1 (E), and ALAS1 (F) were measured by qPCR with the cells used for panel B. Gene expression was normalized to that of the endogenous control GNB2L1 and is shown relative to that of each cell line never treated with HT (Never HT). (G) The H3K4me3 level was assessed by ChIP assay of the LCLs 3CHT shNT, 3CHT shKDM2B-1, and 3CHT shKDM2B-2 used for panel B. ChIP values represent enrichment relative to the input level ± standard deviations for triplicate qPCRs for the ChIP and input of each sample. Data are representative of one of two independent infections.

    Article Snippet: Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam).

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

    Flow cytometry analysis of β1 integrin and α4 integrin expression in control and EBNA 3C-expressing cell lines. Results of staining for control IgG (dotted lines), anti-β1 integrin (black fill), or anti-α4 integrin (gray

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: Flow cytometry analysis of β1 integrin and α4 integrin expression in control and EBNA 3C-expressing cell lines. Results of staining for control IgG (dotted lines), anti-β1 integrin (black fill), or anti-α4 integrin (gray

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques: Flow Cytometry, Cytometry, Expressing, Staining

    EBNA 3C associates with ITGA4 , ITGB1 , ADAM28 , and ADAMDEC1 regulatory regions.

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C associates with ITGA4 , ITGB1 , ADAM28 , and ADAMDEC1 regulatory regions.

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques:

    EBNA 3C binding and histone modification at ITGA4 . (A) EBNA 3 binding at the ITGA4 locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP using anti-H3K27me3

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C binding and histone modification at ITGA4 . (A) EBNA 3 binding at the ITGA4 locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP using anti-H3K27me3

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques: Binding Assay, Modification, Chromatin Immunoprecipitation, Expressing, Stable Transfection

    CXCL10 and CXCL11 are downregulated by EBNA 3C. Real-time PCR analysis of CXCL10 (A and B) and CXCL11 (C and D) mRNA expression in EBV nuclear antigen-expressing cells. Results are shown as means ± standard deviations of three independent experiments

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: CXCL10 and CXCL11 are downregulated by EBNA 3C. Real-time PCR analysis of CXCL10 (A and B) and CXCL11 (C and D) mRNA expression in EBV nuclear antigen-expressing cells. Results are shown as means ± standard deviations of three independent experiments

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques: Real-time Polymerase Chain Reaction, Expressing

    Histone modification at the CXCL gene cluster in EBNA 3C-expressing cells. (A and B) ChIP using anti-histone H3K27me3 antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines analyzed with primers across the

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: Histone modification at the CXCL gene cluster in EBNA 3C-expressing cells. (A and B) ChIP using anti-histone H3K27me3 antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines analyzed with primers across the

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques: Modification, Expressing, Chromatin Immunoprecipitation, Stable Transfection

    EBNA 3C expression reduces chemotaxis of CXCR3-expressing cells. Chemotaxis assays using cell culture supernatant from control or EBNA 3C-expressing cells were performed. Shown is the migration of CXCR3-transfected cells expressed relative to the number

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C expression reduces chemotaxis of CXCR3-expressing cells. Chemotaxis assays using cell culture supernatant from control or EBNA 3C-expressing cells were performed. Shown is the migration of CXCR3-transfected cells expressed relative to the number

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques: Expressing, Chemotaxis Assay, Cell Culture, Migration, Transfection

    EBNA 3C binding and histone modification at ITGB1 . (A) EBNA 3 binding at the ITGB1 locus. The numbers of sequencing reads from EBNA 3-enriched DNA are plotted per million background-subtracted total reads and aligned with the human genome. The positions

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C binding and histone modification at ITGB1 . (A) EBNA 3 binding at the ITGB1 locus. The numbers of sequencing reads from EBNA 3-enriched DNA are plotted per million background-subtracted total reads and aligned with the human genome. The positions

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques: Binding Assay, Modification, Sequencing

    EBNA 3C binding and histone modification at the ADAM gene cluster. (A) EBNA 3 binding at the ADAM gene locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP

    Journal: Journal of Virology

    Article Title: Downregulation of Integrin Receptor-Signaling Genes by Epstein-Barr Virus EBNA 3C via Promoter-Proximal and -Distal Binding Elements

    doi: 10.1128/JVI.07161-11

    Figure Lengend Snippet: EBNA 3C binding and histone modification at the ADAM gene cluster. (A) EBNA 3 binding at the ADAM gene locus. (B) ChIP using anti-EBNA 3C antibodies in control (pz1 and pz3) and EBNA 3C-expressing (E3C-3 and E3C-7) BJAB stable cell lines. (C and D) ChIP

    Article Snippet: ChIP for EBNA 3C and H3K27me3 was carried out as for acetylated histone H3, using 10 μl of sheep polyclonal anti-EBNA 3C antibodies (ab16128; Abcam) or 4 μg of anti-H3K27me3 antibodies (17-622; Millipore), respectively.

    Techniques: Binding Assay, Modification, Chromatin Immunoprecipitation, Expressing, Stable Transfection

    This figure represents the details of the 3C/LIC cloning strategy, PCR product containing the 5LIC extension were treated with LIC-qualified T4 DNA polymerase in the presence of dATB, and then annealed to the 3C/LIC vector.

    Journal: Saudi Journal of Biological Sciences

    Article Title: Expression of variable viruses as herpes simplex glycoprotein D and varicella zoster gE glycoprotein using a novel plasmid based expression system in insect cell

    doi: 10.1016/j.sjbs.2016.05.003

    Figure Lengend Snippet: This figure represents the details of the 3C/LIC cloning strategy, PCR product containing the 5LIC extension were treated with LIC-qualified T4 DNA polymerase in the presence of dATB, and then annealed to the 3C/LIC vector.

    Article Snippet: Briefly, the insert with overhangs prepared by PCR was treated with T4 DNA polymerase in the presence of dATP, and then annealed with the pIEx/Bac-3 3C/LIC vector (Novagen, Merck, USA) containing the hr5 (homologous region 5) enhancer and the ie1 (immediate early 1) promoter from Autographa californica nuclear polyhedrosis virus (AcNPV).

    Techniques: Clone Assay, Polymerase Chain Reaction, Plasmid Preparation

    Construction of the recombinant plasmid pcDNA3.1/P12A3C. PCR ( A ), enzyme digestion ( B ). Notes: M1 and M2 indicates DNA ladder markers; Lane 1 indicates the PCR product of P12A; lane 2 indicates the PCR product of 3C; lane 3 indicates EcoR I- and Xbal I-digested pcDNA3.1/P12A3C; lane 4 indicates BamH I- and Xbal I-digested; lane 5 indicates BamH I- and EcoR I-digested. Abbreviation: PCR, polymerase chain reaction.

    Journal: International Journal of Nanomedicine

    Article Title: Induction of mucosal immune responses and protection of cattle against direct-contact challenge by intranasal delivery with foot-and-mouth disease virus antigen mediated by nanoparticles

    doi: 10.2147/IJN.S72318

    Figure Lengend Snippet: Construction of the recombinant plasmid pcDNA3.1/P12A3C. PCR ( A ), enzyme digestion ( B ). Notes: M1 and M2 indicates DNA ladder markers; Lane 1 indicates the PCR product of P12A; lane 2 indicates the PCR product of 3C; lane 3 indicates EcoR I- and Xbal I-digested pcDNA3.1/P12A3C; lane 4 indicates BamH I- and Xbal I-digested; lane 5 indicates BamH I- and EcoR I-digested. Abbreviation: PCR, polymerase chain reaction.

    Article Snippet: P12A was digested using BamH I and EcoR I, and 3C was digested with EcoR I and Xbal I, and fragments were subcloned into the corresponding sites of pcDNA3.1(+) (Invitrogen® ; Life Technologies) using T4 DNA ligase to generate recombinant plasmids pcDNA3.1/P12A3C.

    Techniques: Recombinant, Plasmid Preparation, Polymerase Chain Reaction

    3C analysis indicates contacts between the PLA2G4A and COX-2 promoters. The 3C experiments were performed using mismatch (MM)- or RNA12-treated sample (25 nM). ( A ) Diagram of COX-2/PLA2G4A loci and their intergenic region. Restriction enzyme Dpn II was used to digest chromosomal DNAs. Restriction sites for Dpn II and location of 3C primers are depicted by triangles and arrows, respectively. The 3C PCRs were performed using combinations of a constant primer (C1 or C2) and test primers (T1–8). Primer C1 and C2 are located within the constant fragment (−1077 to −354), which contains the COX-2 promoter sequence. Primers T4, T5 and T6 target the PLA2G4A promoter (∼149k bases). ( B ) Analysis of 3C PCR products on 2.5% agarose gels (C1 + T4, C1 + T5). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( C ) Analysis of 3C PCR products on 2.5% agarose gels (C2 + T1–8). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( D ) Quantitative analysis of relative cross-linking frequencies using PrimeTime qPCR assay. n = 4. Error bars are SEM. * P

    Journal: Nucleic Acids Research

    Article Title: Promoter RNA links transcriptional regulation of inflammatory pathway genes

    doi: 10.1093/nar/gkt777

    Figure Lengend Snippet: 3C analysis indicates contacts between the PLA2G4A and COX-2 promoters. The 3C experiments were performed using mismatch (MM)- or RNA12-treated sample (25 nM). ( A ) Diagram of COX-2/PLA2G4A loci and their intergenic region. Restriction enzyme Dpn II was used to digest chromosomal DNAs. Restriction sites for Dpn II and location of 3C primers are depicted by triangles and arrows, respectively. The 3C PCRs were performed using combinations of a constant primer (C1 or C2) and test primers (T1–8). Primer C1 and C2 are located within the constant fragment (−1077 to −354), which contains the COX-2 promoter sequence. Primers T4, T5 and T6 target the PLA2G4A promoter (∼149k bases). ( B ) Analysis of 3C PCR products on 2.5% agarose gels (C1 + T4, C1 + T5). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( C ) Analysis of 3C PCR products on 2.5% agarose gels (C2 + T1–8). The internal region of the constant fragment was amplified using primer set −740/−600 as a control. In vitro -synthesized DNA templates that have sequences of expected ligation products were used as positive control and samples without cross-linking were used as negative control. ( D ) Quantitative analysis of relative cross-linking frequencies using PrimeTime qPCR assay. n = 4. Error bars are SEM. * P

    Article Snippet: The 3C products were further purified using Wizard SV Gel and PCR clean-up system (Promega).

    Techniques: Sequencing, Polymerase Chain Reaction, Amplification, In Vitro, Synthesized, Ligation, Positive Control, Negative Control, Real-time Polymerase Chain Reaction

    Biochemical characterization of hMex-3 proteins. ( A and B ) BOSC cells were transiently transfected with vectors expressing myc-tagged forms of hMex-3A, -3B and -3C. Western blot analysis was performed with anti-myc antibody. In (B) treatment of protein extracts with (+) or without (−) λ-Phosphatase. ( C ) Kinase assay. hMex-3A and -3B proteins or a control protein (Bpag1) expressed in BOSC cells were immunoprecipitated with the anti-myc antibody and incubated with kinase buffer and [γ 32 P] ATP. Labeled proteins were revealed by autoradiography. ( D ) RNA homopolymer binding assay. Proteins from indicated expression vectors were in vitro translated in the presence of [ 35 S] methionine ( top ). Binding to agarose beads coupled to poly(A) ( bottom ) RNA homopolymers is shown for in vitro translated proteins. As a negative control, a fragment of P62-sequestosome protein was incubated with RNA homopolymers in the same conditions. One-tenth of the initial translation reactions and all the bound proteins were analysed by SDS-PAGE and autoradiography. ( E ) In vivo hMex-3 binding to mRNA. BOSC cells were transiently transfected with vectors expressing myc-tagged proteins, as indicated. RT-PCR amplification was performed on total RNA extracted from those cells ( top left ). Western blot analysis performed with anti-myc antibody ( bottom left ). RT-PCR amplification performed on total RNA extracted from sepharose-protein A beads after immunoprecipitation by an anti-myc antibody ( right ).

    Journal: Nucleic Acids Research

    Article Title: Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies

    doi: 10.1093/nar/gkm016

    Figure Lengend Snippet: Biochemical characterization of hMex-3 proteins. ( A and B ) BOSC cells were transiently transfected with vectors expressing myc-tagged forms of hMex-3A, -3B and -3C. Western blot analysis was performed with anti-myc antibody. In (B) treatment of protein extracts with (+) or without (−) λ-Phosphatase. ( C ) Kinase assay. hMex-3A and -3B proteins or a control protein (Bpag1) expressed in BOSC cells were immunoprecipitated with the anti-myc antibody and incubated with kinase buffer and [γ 32 P] ATP. Labeled proteins were revealed by autoradiography. ( D ) RNA homopolymer binding assay. Proteins from indicated expression vectors were in vitro translated in the presence of [ 35 S] methionine ( top ). Binding to agarose beads coupled to poly(A) ( bottom ) RNA homopolymers is shown for in vitro translated proteins. As a negative control, a fragment of P62-sequestosome protein was incubated with RNA homopolymers in the same conditions. One-tenth of the initial translation reactions and all the bound proteins were analysed by SDS-PAGE and autoradiography. ( E ) In vivo hMex-3 binding to mRNA. BOSC cells were transiently transfected with vectors expressing myc-tagged proteins, as indicated. RT-PCR amplification was performed on total RNA extracted from those cells ( top left ). Western blot analysis performed with anti-myc antibody ( bottom left ). RT-PCR amplification performed on total RNA extracted from sepharose-protein A beads after immunoprecipitation by an anti-myc antibody ( right ).

    Article Snippet: To check expression level of the four hMex -3 genes, Cdx 2 gene or GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and β2M (β2-microglobulin) genes as controls, 40 cycles (35 for Cdx 2, 30 for GAPDH and β2M ) of PCR were performed according to the manufacturer's instructions on cDNA with Taq DNA Polymerase (Invitrogen) for GAPDH and β2M , High Fidelity PCR system (Roche), for hMex -3C or GC-rich PCR system (Roche) for hMex -3A, hMex -3B, hMex -3D and Cdx 2 in presence of 200 nM internal primers specific for each cDNA: hM3AiF:5′-AGGGCTGCTGGAAGACGAG-3′, hM3AiR:5′-TGAGATGATTTCCCGCCG-3′, hM3BiF:5′-CCTGCTGGGGCTGGACA-3′, hM3BiR:5′-TGGAAGTCGTTCTCGTCTGT-3′, hM3CiF:5′-TGAACGGGGAGCAGGCG-3′, hM3CiR:5′-TGACTTGGACGGTGGTTTGA-3′, hM3DiF:5′-CCGACCAGATGAGCGTGAT-3′, hM3DiR:5′-CGAGCAGGTCCAGGCAGA-3′, Cdx2Fi:5′-GAACCTGTGCGAGTGGATG-3′, Cdx2Ri:5′-TCTCAGAGGACCTGGCTGAG-3′, GAPDHiF:5′-TCCCATCACCATCTTCCAGG-3′, GAPDHiR:5′-ATGAGTCCTTCCACGATACC-3′, β2MF:5′-TCGCGCTACTCTCTCTTTCTG-3′, β2MR:5′-AACTTCAATGTCGGATGGATG-3′.

    Techniques: Transfection, Expressing, Western Blot, Kinase Assay, Immunoprecipitation, Incubation, Labeling, Autoradiography, Binding Assay, In Vitro, Negative Control, SDS Page, In Vivo, Reverse Transcription Polymerase Chain Reaction, Amplification

    Expression profile of hMex -3 genes and hMex-3B protein. ( A ) Human Mex -3 gene expression levels ( panels 1–4 ) were examined by RT-PCR with specific internal primers and were compared with expression level of ubiquitously expressed GAPDH gene ( panel 5 ). RNA were extracted from 7 human cell lines ( left ) and from 20 human tissues (Multiple Tissue Total RNA panel, BD Biosciences) ( right ). ( B ) Human colon sections (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ antibody ( panels 1 and 2 ), anti-hMex-3Bβ antibody + hMex-3B peptide, as control ( panel 3 ). ( C ) Serial sections of human Meckel's diverticulum (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ ( top left ) and anti-MUC2 ( bottom left ), anti-hMex-3Bβ ( top middle ) and anti-Chromogranin-A ( bottom middle ), anti-hMex-3Bβ ( top right ) and anti-Lysozyme ( bottom right ).

    Journal: Nucleic Acids Research

    Article Title: Identification and characterization of human Mex-3 proteins, a novel family of evolutionarily conserved RNA-binding proteins differentially localized to processing bodies

    doi: 10.1093/nar/gkm016

    Figure Lengend Snippet: Expression profile of hMex -3 genes and hMex-3B protein. ( A ) Human Mex -3 gene expression levels ( panels 1–4 ) were examined by RT-PCR with specific internal primers and were compared with expression level of ubiquitously expressed GAPDH gene ( panel 5 ). RNA were extracted from 7 human cell lines ( left ) and from 20 human tissues (Multiple Tissue Total RNA panel, BD Biosciences) ( right ). ( B ) Human colon sections (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ antibody ( panels 1 and 2 ), anti-hMex-3Bβ antibody + hMex-3B peptide, as control ( panel 3 ). ( C ) Serial sections of human Meckel's diverticulum (magnification: ×100 or ×400) were stained with anti-hMex-3Bβ ( top left ) and anti-MUC2 ( bottom left ), anti-hMex-3Bβ ( top middle ) and anti-Chromogranin-A ( bottom middle ), anti-hMex-3Bβ ( top right ) and anti-Lysozyme ( bottom right ).

    Article Snippet: To check expression level of the four hMex -3 genes, Cdx 2 gene or GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and β2M (β2-microglobulin) genes as controls, 40 cycles (35 for Cdx 2, 30 for GAPDH and β2M ) of PCR were performed according to the manufacturer's instructions on cDNA with Taq DNA Polymerase (Invitrogen) for GAPDH and β2M , High Fidelity PCR system (Roche), for hMex -3C or GC-rich PCR system (Roche) for hMex -3A, hMex -3B, hMex -3D and Cdx 2 in presence of 200 nM internal primers specific for each cDNA: hM3AiF:5′-AGGGCTGCTGGAAGACGAG-3′, hM3AiR:5′-TGAGATGATTTCCCGCCG-3′, hM3BiF:5′-CCTGCTGGGGCTGGACA-3′, hM3BiR:5′-TGGAAGTCGTTCTCGTCTGT-3′, hM3CiF:5′-TGAACGGGGAGCAGGCG-3′, hM3CiR:5′-TGACTTGGACGGTGGTTTGA-3′, hM3DiF:5′-CCGACCAGATGAGCGTGAT-3′, hM3DiR:5′-CGAGCAGGTCCAGGCAGA-3′, Cdx2Fi:5′-GAACCTGTGCGAGTGGATG-3′, Cdx2Ri:5′-TCTCAGAGGACCTGGCTGAG-3′, GAPDHiF:5′-TCCCATCACCATCTTCCAGG-3′, GAPDHiR:5′-ATGAGTCCTTCCACGATACC-3′, β2MF:5′-TCGCGCTACTCTCTCTTTCTG-3′, β2MR:5′-AACTTCAATGTCGGATGGATG-3′.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining

    Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control PCR panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 qRT-PCR at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).

    Journal: Molecular Cell

    Article Title: Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals

    doi: 10.1016/j.molcel.2007.11.030

    Figure Lengend Snippet: Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control PCR panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 qRT-PCR at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).

    Article Snippet: Real-time (qRT-PCR) 3C used 2 μl of input in triplicate (Corbett Rotor-Gene 3000 with QuantiTect SYBR Green [QIAGEN)).

    Techniques: Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Amplification

    Characterization of Integrated HIV-1 Provirus in U1 Cells (A) (a) HIV-1 mRNA accumulation using qRT-PCR analysis. Values were normalized to 18S rRNA and expressed as a fold induction over untreated U1 cells. (b) Location of ChIP primers relative to the transcription start site (+1) are depicted. Nuc, nucleosomes present on the 5′LTR of latent provirus. Note: P1 does not distinguish between the two LTRs. (c) Pol II qChIP on control (white bars) and TPA-activated U1 cells after 5 (black bars) and 24 hr (gray bars) using the indicated primer and probe sets. Binding was measured relative to B13 (see Experimental Procedures ). Error bars represent SEM from n = 3 samples performed in duplicate for each primer set. (B) HIV-1 proviral characterization using iPCR. (a) iPCR primer locations and restriction sites: gray, outer primers; black, nested primers. Open arrows illustrate 3′ HIV-proviral/host cell DNA junctions used to confirm integrate location (see Experimental Procedures ). (b) Nested U1 genomic DNA PCR products (+; with Taq polymerase) for integration site mapping and determination of Tat sequences for int-Chr2 (Chr2; Tat1 U1 ) and int-ChrX (ChrX; Tat2 U1 ).

    Journal: Molecular Cell

    Article Title: Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals

    doi: 10.1016/j.molcel.2007.11.030

    Figure Lengend Snippet: Characterization of Integrated HIV-1 Provirus in U1 Cells (A) (a) HIV-1 mRNA accumulation using qRT-PCR analysis. Values were normalized to 18S rRNA and expressed as a fold induction over untreated U1 cells. (b) Location of ChIP primers relative to the transcription start site (+1) are depicted. Nuc, nucleosomes present on the 5′LTR of latent provirus. Note: P1 does not distinguish between the two LTRs. (c) Pol II qChIP on control (white bars) and TPA-activated U1 cells after 5 (black bars) and 24 hr (gray bars) using the indicated primer and probe sets. Binding was measured relative to B13 (see Experimental Procedures ). Error bars represent SEM from n = 3 samples performed in duplicate for each primer set. (B) HIV-1 proviral characterization using iPCR. (a) iPCR primer locations and restriction sites: gray, outer primers; black, nested primers. Open arrows illustrate 3′ HIV-proviral/host cell DNA junctions used to confirm integrate location (see Experimental Procedures ). (b) Nested U1 genomic DNA PCR products (+; with Taq polymerase) for integration site mapping and determination of Tat sequences for int-Chr2 (Chr2; Tat1 U1 ) and int-ChrX (ChrX; Tat2 U1 ).

    Article Snippet: Real-time (qRT-PCR) 3C used 2 μl of input in triplicate (Corbett Rotor-Gene 3000 with QuantiTect SYBR Green [QIAGEN)).

    Techniques: Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Polymerase Chain Reaction

    R1881-induction of SEMA3C expression is GATA2-dependent We examined R1881-induced expression of SEMA3C in the absence of GATA2 to confirm findings from previous microarray studies showing that knockdown of GATA2 decreases SEMA3C expression. When compared to LNCaP treated with scrambled siRNA (siSCX), knockdown of GATA2 (siGATA2) triggered a significant decrease in basal SEMA3C expression and completely attenuated R1881-mediated dose-dependent induction of SEMA3C as shown by qPCR A . These observations were confirmed at the protein level by Western blot analysis B . of both conditioned media (CM) and whole cell extract (WCE) where total actin served as loading control. In chromatin immunoprecipitation assays, SEMA3C ARE amplicon was shown be enriched in GATA2 immunoprecipitates of lysates from LNCaP cells treated with R1881 as shown by end-point C . and qPCR D . indicating an R1881-dependent recruitment of GATA2 to the SEMA3C intron 2 ARE. Input = 1% input, Isotype = isotype-matched control antibody, IP: GATA2 = GATA2 immunoprecipitates. PCR primers for these experiments were the same as those for Figure 3 and map to the SEMA3C intron 2 ARE. ChIP qPCR values represent a fold enrichment over isotype control of the same treatment condition. Chromatin immunoprecipitation assays previously showing R1881-induced recruitment of AR to the SEMA3C ARE were repeated in the presence of siRNA to GATA2 E . Values represent a fold enrichment over isotype control of the same treatment condition. Data represent mean, ± SD; * p

    Journal: Oncotarget

    Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner

    doi: 10.18632/oncotarget.14168

    Figure Lengend Snippet: R1881-induction of SEMA3C expression is GATA2-dependent We examined R1881-induced expression of SEMA3C in the absence of GATA2 to confirm findings from previous microarray studies showing that knockdown of GATA2 decreases SEMA3C expression. When compared to LNCaP treated with scrambled siRNA (siSCX), knockdown of GATA2 (siGATA2) triggered a significant decrease in basal SEMA3C expression and completely attenuated R1881-mediated dose-dependent induction of SEMA3C as shown by qPCR A . These observations were confirmed at the protein level by Western blot analysis B . of both conditioned media (CM) and whole cell extract (WCE) where total actin served as loading control. In chromatin immunoprecipitation assays, SEMA3C ARE amplicon was shown be enriched in GATA2 immunoprecipitates of lysates from LNCaP cells treated with R1881 as shown by end-point C . and qPCR D . indicating an R1881-dependent recruitment of GATA2 to the SEMA3C intron 2 ARE. Input = 1% input, Isotype = isotype-matched control antibody, IP: GATA2 = GATA2 immunoprecipitates. PCR primers for these experiments were the same as those for Figure 3 and map to the SEMA3C intron 2 ARE. ChIP qPCR values represent a fold enrichment over isotype control of the same treatment condition. Chromatin immunoprecipitation assays previously showing R1881-induced recruitment of AR to the SEMA3C ARE were repeated in the presence of siRNA to GATA2 E . Values represent a fold enrichment over isotype control of the same treatment condition. Data represent mean, ± SD; * p

    Article Snippet: Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505).

    Techniques: Expressing, Microarray, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Amplification, Polymerase Chain Reaction

    SEMA3C is an androgen receptor-regulated gene LNCaP were treated with increasing concentrations (0 to 5 nM) of the synthetic androgen, R1881 A ., and increasing concentrations (0 to 10 nM) of dihydrotestosterone (DHT; B .) followed by detection of SEMA3C mRNA levels by qPCR; relative quantities (RQ) are presented. R1881-stimulated LNCaP cells were treated with increasing concentrations (6.25, 12.5, 25, and 50 μM) of Enzalutamide (MDV3100; C .) or of the AR DBD inhibitor VPC-14449 (14449; D .) followed by SEMA3C mRNA level detection by qPCR. AR-negative PCa lines PC-3 and DU 145 did not upregulate SEMA3C in response to R1881 E F . PC-3 and LNCaP cells were transfected with mock or AR overexpression plasmids followed by qPCR for SEMA3C G H . LNCaP cells were transfected with siRNA directed against AR (siAR) or scrambled siRNA (siSCX) followed by qPCR for SEMA3C I. LNCaP cells were treated with R1881 (5 nM) in the presence of siAR followed by qPCR for SEMA3C J . LNCaP were treated with PI3K inhibitor LY294002 at the indicated concentrations or DMSO and monitored for SEMA3C message expression K . LNCaP were treated with LY294002 (40 μM) in the presence of siRNA for AR (siAR) or GATA2 (siGATA2) followed by qPCR for SEMA3C L . 22Rv1 were treated with PTEN inhibitor bpV(HOpic) at the indicated concentrations or DMSO and monitored for SEMA3C message expression M . Data represent mean, ± SD; * p

    Journal: Oncotarget

    Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner

    doi: 10.18632/oncotarget.14168

    Figure Lengend Snippet: SEMA3C is an androgen receptor-regulated gene LNCaP were treated with increasing concentrations (0 to 5 nM) of the synthetic androgen, R1881 A ., and increasing concentrations (0 to 10 nM) of dihydrotestosterone (DHT; B .) followed by detection of SEMA3C mRNA levels by qPCR; relative quantities (RQ) are presented. R1881-stimulated LNCaP cells were treated with increasing concentrations (6.25, 12.5, 25, and 50 μM) of Enzalutamide (MDV3100; C .) or of the AR DBD inhibitor VPC-14449 (14449; D .) followed by SEMA3C mRNA level detection by qPCR. AR-negative PCa lines PC-3 and DU 145 did not upregulate SEMA3C in response to R1881 E F . PC-3 and LNCaP cells were transfected with mock or AR overexpression plasmids followed by qPCR for SEMA3C G H . LNCaP cells were transfected with siRNA directed against AR (siAR) or scrambled siRNA (siSCX) followed by qPCR for SEMA3C I. LNCaP cells were treated with R1881 (5 nM) in the presence of siAR followed by qPCR for SEMA3C J . LNCaP were treated with PI3K inhibitor LY294002 at the indicated concentrations or DMSO and monitored for SEMA3C message expression K . LNCaP were treated with LY294002 (40 μM) in the presence of siRNA for AR (siAR) or GATA2 (siGATA2) followed by qPCR for SEMA3C L . 22Rv1 were treated with PTEN inhibitor bpV(HOpic) at the indicated concentrations or DMSO and monitored for SEMA3C message expression M . Data represent mean, ± SD; * p

    Article Snippet: Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505).

    Techniques: Real-time Polymerase Chain Reaction, Transfection, Over Expression, Expressing

    The androgen receptor associates with the SEMA3C intron 2 ARE In electrophoretic mobility shift assays, 50 basepair oligonucleotides at a final concentration of 1.875 μM were combined with increasing concentrations (0, 0.5, 1.0, 2.0 μM) of purified human androgen receptor DNA-binding domain (AR DBD) and run on a non-denaturing acrylamide gel. A shift (at arrow) was observed when AR DBD was combined with oligonucleotide containing the intron 2 ARE (wtARE) but not when combined with either a 50 bp oligonucleotide mapping to ∼200 bp downstream of the intron 2 ARE (downARE) A . nor when combined with an oligonucleotide with transversion mutations to six bases of the ARE (mutARE) B . Sequences of the oligonucleotides used for the assay are shown below; sequences shown are complementary to those of Figure 1 : Bases matching the JASPAR motif are shown in colour; mutations are underlined. ChIP assays were carried out on lysates of LNCaP treated with 0.05% ethanol (vehicle control) or 5 nM R1881. PCR was performed on 1% input (Input), isotype-matched control (Isotype), and AR immunoprecipitates (IP: AR). C . End-point PCR showed abundant levels of SEMA3C ARE amplicon in input and undetectable levels in isotype control irrespective of R1881 treatment. Ethanol-treated AR immunoprecipitates showed low but detectable levels of SEMA3C ARE amplicon whereas R1881 triggered enriched SEMA3C ARE amplicon in AR immunoprecipitates. Results were confirmed by qPCR D .; values represent fold enrichment over isotype control of the same treatment condition. ± SD; ** p

    Journal: Oncotarget

    Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner

    doi: 10.18632/oncotarget.14168

    Figure Lengend Snippet: The androgen receptor associates with the SEMA3C intron 2 ARE In electrophoretic mobility shift assays, 50 basepair oligonucleotides at a final concentration of 1.875 μM were combined with increasing concentrations (0, 0.5, 1.0, 2.0 μM) of purified human androgen receptor DNA-binding domain (AR DBD) and run on a non-denaturing acrylamide gel. A shift (at arrow) was observed when AR DBD was combined with oligonucleotide containing the intron 2 ARE (wtARE) but not when combined with either a 50 bp oligonucleotide mapping to ∼200 bp downstream of the intron 2 ARE (downARE) A . nor when combined with an oligonucleotide with transversion mutations to six bases of the ARE (mutARE) B . Sequences of the oligonucleotides used for the assay are shown below; sequences shown are complementary to those of Figure 1 : Bases matching the JASPAR motif are shown in colour; mutations are underlined. ChIP assays were carried out on lysates of LNCaP treated with 0.05% ethanol (vehicle control) or 5 nM R1881. PCR was performed on 1% input (Input), isotype-matched control (Isotype), and AR immunoprecipitates (IP: AR). C . End-point PCR showed abundant levels of SEMA3C ARE amplicon in input and undetectable levels in isotype control irrespective of R1881 treatment. Ethanol-treated AR immunoprecipitates showed low but detectable levels of SEMA3C ARE amplicon whereas R1881 triggered enriched SEMA3C ARE amplicon in AR immunoprecipitates. Results were confirmed by qPCR D .; values represent fold enrichment over isotype control of the same treatment condition. ± SD; ** p

    Article Snippet: Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505).

    Techniques: Electrophoretic Mobility Shift Assay, Concentration Assay, Purification, Binding Assay, Acrylamide Gel Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Androgen receptor transactivates the SEMA3C intron 2 ARE A . LNCaP cells were transfected with empty pGL3-Basic reporter plasmid (Basic), SEMA3C intron 2 ARE reporter plasmids (wtARE), or reporter plasmids where the ARE was mutated (mutARE). Sequences cloned into reporter plasmids are shown below; sequences shown are complementary to those of Figure 1 : Bases matching the JASPAR motif are shown in colour and ARE and GATA2 elements are indicated; mutations are underlined. Cells were then treated with R1881 at 5 nM or vehicle control (0.05% EtOH) and harvested for measurement of relative luminescence (RLU). B . 293T were co-transfected with reporter plasmids (Basic or wtARE) and AR overexpression plasmids followed by treatment with R1881 at 1 nM or vehicle. C . LNCaP cells were transfected with wtARE, wtARE with the final 60 basepairs truncated (wtARE-60bp), wtARE with the final 120 basepair truncated (wtARE-120bp), and Basic (sequences are shown below). The wtARE construct contains both the AR and GATA2 motif; the wtARE-60bp construct has the GATA2 motif removed but retains the AR motif; the wtARE-120bp construct contains neither the AR nor the GATA2 motif. Transfected cells were then treated with R1881 at 5 nM or vehicle. D . 293T cells were transfected with wtARE and either wtAR or ARv7 overexpression plasmid and subsequently treated with R1881 to 1 nM. Transfection with pcDNA3.1 served as a control. Values represent a fold increase over EtOH control (A-C) or fold increase over pcDNA3.1/EtOH treatment (D). Data represent mean, ± SD; * p

    Journal: Oncotarget

    Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner

    doi: 10.18632/oncotarget.14168

    Figure Lengend Snippet: Androgen receptor transactivates the SEMA3C intron 2 ARE A . LNCaP cells were transfected with empty pGL3-Basic reporter plasmid (Basic), SEMA3C intron 2 ARE reporter plasmids (wtARE), or reporter plasmids where the ARE was mutated (mutARE). Sequences cloned into reporter plasmids are shown below; sequences shown are complementary to those of Figure 1 : Bases matching the JASPAR motif are shown in colour and ARE and GATA2 elements are indicated; mutations are underlined. Cells were then treated with R1881 at 5 nM or vehicle control (0.05% EtOH) and harvested for measurement of relative luminescence (RLU). B . 293T were co-transfected with reporter plasmids (Basic or wtARE) and AR overexpression plasmids followed by treatment with R1881 at 1 nM or vehicle. C . LNCaP cells were transfected with wtARE, wtARE with the final 60 basepairs truncated (wtARE-60bp), wtARE with the final 120 basepair truncated (wtARE-120bp), and Basic (sequences are shown below). The wtARE construct contains both the AR and GATA2 motif; the wtARE-60bp construct has the GATA2 motif removed but retains the AR motif; the wtARE-120bp construct contains neither the AR nor the GATA2 motif. Transfected cells were then treated with R1881 at 5 nM or vehicle. D . 293T cells were transfected with wtARE and either wtAR or ARv7 overexpression plasmid and subsequently treated with R1881 to 1 nM. Transfection with pcDNA3.1 served as a control. Values represent a fold increase over EtOH control (A-C) or fold increase over pcDNA3.1/EtOH treatment (D). Data represent mean, ± SD; * p

    Article Snippet: Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505).

    Techniques: Transfection, Plasmid Preparation, Clone Assay, Over Expression, Construct

    FOXA1 negatively regulates SEMA3C expression We assessed the effect of silencing of FOXA1 and POU2F1 on SEMA3C message levels. A . Knockdown of FOXA1 (siFOXA1) triggered an increase in SEMA3C levels when compared to cells treated with scrambled siRNA (siSCX); knockdown of POU2F1 (siPOU2F1) had no effect on SEMA3C expression. Knockdown of these genes was confirmed by qPCR (POU2F1) or Western blot (FOXA1; B .). In siFOXA1-treated cells where SEMA3C levels were already elevated, SEMA3C expression was further increased upon R1881 stimulation shown at both the message C . and protein D . level. Knockdown of POU2F1 had little effect on R1881 induction of SEMA3C (C, D). WCE: whole cell extract; CM: conditioned media. E . LNCaP cells were knocked down with siAR, siFOXA1, or both siAR and siFOXA1 and monitored for SEMA3C expression. Compared to LNCaP treated with scrambled siRNA, siAR-treated cells had decreased SEMA3C expression while siFOXA1 and siAR+siFOXA1-treated cells had elevated SEMA3C expression. Knockdown of AR and FOXA1 was confirmed by Western blot analysis (E). Data represent mean, ± SD; ** p

    Journal: Oncotarget

    Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner

    doi: 10.18632/oncotarget.14168

    Figure Lengend Snippet: FOXA1 negatively regulates SEMA3C expression We assessed the effect of silencing of FOXA1 and POU2F1 on SEMA3C message levels. A . Knockdown of FOXA1 (siFOXA1) triggered an increase in SEMA3C levels when compared to cells treated with scrambled siRNA (siSCX); knockdown of POU2F1 (siPOU2F1) had no effect on SEMA3C expression. Knockdown of these genes was confirmed by qPCR (POU2F1) or Western blot (FOXA1; B .). In siFOXA1-treated cells where SEMA3C levels were already elevated, SEMA3C expression was further increased upon R1881 stimulation shown at both the message C . and protein D . level. Knockdown of POU2F1 had little effect on R1881 induction of SEMA3C (C, D). WCE: whole cell extract; CM: conditioned media. E . LNCaP cells were knocked down with siAR, siFOXA1, or both siAR and siFOXA1 and monitored for SEMA3C expression. Compared to LNCaP treated with scrambled siRNA, siAR-treated cells had decreased SEMA3C expression while siFOXA1 and siAR+siFOXA1-treated cells had elevated SEMA3C expression. Knockdown of AR and FOXA1 was confirmed by Western blot analysis (E). Data represent mean, ± SD; ** p

    Article Snippet: Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot

    ARE and GATA2 DNA motifs at the human SEMA3C locus A . The ChIP-Seq peaks (vertical black bars) from Yu et al were overlaid on the SEMA3C locus (blue horizontal line: exons are shown as vertical blue bars) in the UCSC Genome Browser (hg18). A red horizontal line illustrates the average peak height from the ChIP-Seq experiment. The DNA sequences containing the ARE and GATA2 motifs, as predicted by the Patser program, are shown within the two peaks that span from 80,351,826 to 80,352,350 in intron 2 and from 80,248,676 to 80,249,175 in intron 12, respectively. B . A consensus GATA2 motif documented in the JASPAR database is illustrated as a sequence logo. C . A consensus ARE motif documented in the JASPAR database is illustrated as a sequence logo.

    Journal: Oncotarget

    Article Title: Androgen receptor transcriptionally regulates semaphorin 3C in a GATA2-dependent manner

    doi: 10.18632/oncotarget.14168

    Figure Lengend Snippet: ARE and GATA2 DNA motifs at the human SEMA3C locus A . The ChIP-Seq peaks (vertical black bars) from Yu et al were overlaid on the SEMA3C locus (blue horizontal line: exons are shown as vertical blue bars) in the UCSC Genome Browser (hg18). A red horizontal line illustrates the average peak height from the ChIP-Seq experiment. The DNA sequences containing the ARE and GATA2 motifs, as predicted by the Patser program, are shown within the two peaks that span from 80,351,826 to 80,352,350 in intron 2 and from 80,248,676 to 80,249,175 in intron 12, respectively. B . A consensus GATA2 motif documented in the JASPAR database is illustrated as a sequence logo. C . A consensus ARE motif documented in the JASPAR database is illustrated as a sequence logo.

    Article Snippet: Primary antibodies: phospho-Akt (Ser473; Cell Signaling Technology, Cat. No. 4060S), pan-Akt, (Life Technologies, Cat. No. 44609G), androgen receptor (Santa Cruz Biotechnology, Cat. No. sc-816), SEMA3C (Santa Cruz Biotechnology, Cat. No. sc-27796), GATA2 (Santa Cruz Biotechnology, Cat. No. sc-9008), FOXA1 (Santa Cruz Biotechnology, Cat. No. sc-6553), POU2F1 (Santa Cruz Biotechnology, Cat. No.s sc-232, sc-8024; Cell Signaling Technology, Cat. No. 4428S), actin (Sigma-Aldrich, Cat. No. A2066), and vinculin (Sigma-Aldrich, Cat. No. V4505).

    Techniques: Chromatin Immunoprecipitation, Sequencing