thermostable fen1  (New England Biolabs)


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    Structured Review

    New England Biolabs thermostable fen1
    The <t>FEN1</t> inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P
    Thermostable Fen1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermostable fen1/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    thermostable fen1 - by Bioz Stars, 2022-05
    93/100 stars

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    1) Product Images from "Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus"

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007124

    The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P
    Figure Legend Snippet: The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Techniques Used: Inhibition, Real-time Polymerase Chain Reaction

    Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P
    Figure Legend Snippet: Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Techniques Used: Activity Assay, Mutagenesis, Immunoprecipitation, shRNA, Selection, Southern Blot, Real-time Polymerase Chain Reaction

    Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Cotransfection, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P
    Figure Legend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Techniques Used: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P
    Figure Legend Snippet: FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Techniques Used: CRISPR, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Modification

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    New England Biolabs thermostable fen1
    The <t>FEN1</t> inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P
    Thermostable Fen1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/thermostable fen1/product/New England Biolabs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    thermostable fen1 - by Bioz Stars, 2022-05
    93/100 stars
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    The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: The FEN1 inhibitor, PTPD, reduces cccDNA production. Effect of FEN1 inhibition on HBV-replicating cells. Hep38.7-Tet cells were treated with dimethylsulfoxide (DMSO) as a vehicle control, PTPD (5 μM), or 3TC (50 μM) in the absence of tetracycline for 5 days. At day 5, levels of HBV DNA, HBV RNA (pgRNA normalized by HPRT) and pre-C mRNA were analyzed. qPCR analysis of HBV DNA in (A) culture supernatant, (B) cytoplasmic NC-DNA, (C) cccDNA, and (D) pgRNA. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).

    Techniques: Inhibition, Real-time Polymerase Chain Reaction

    Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: Requirement of nuclease activity and the C-terminus of FEN1 for cccDNA production. (A) Schematic presentation of FEN1 protein. D181A: nuclease-deficient mutant. ΔC: deletion mutant unable to bind WRN protein. (B) FEN assay. Flap endonuclease activity of immunoprecipitated FEN1 protein (wt, D181A, or ΔC) was determined as in Fig 1A . (C) pResQ lentiviral vectors carrying FEN1 shRNA and the FEN1 transgene (wt, D181A, or ΔC, see also S8 Fig ) were transduced into Hep38.7-Tet cells. After puromycin selection, NC-DNA was analyzed by Southern blotting and qPCR, respectively. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; **** P

    Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).

    Techniques: Activity Assay, Mutagenesis, Immunoprecipitation, shRNA, Selection, Southern Blot, Real-time Polymerase Chain Reaction

    Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: Deletion of the C-terminus disrupts the nuclear localization and reduces HBV DNA association of FEN1 protein. (A) Expression vectors of FEN1-GFP, FEN1ΔC-GFP, or mock vector (pcDNA4/TO) were transfected into Hep38.7-Tet cells. The nucleus was visualized by co-transfection of the nuclear localization signal (NLS)-tagged DsRed vector. (B–C) Myc-FEN1 or Myc-FEN1ΔC vector was transfected into Hep38.7-Tet cells. (B) Myc-tagged protein expression (before cross linkage) shown by Western blot. Two blots with different protein loadings are shown. (C) Myc-FEN1-transfected Hep38.7-Tet cells were cross-linked, and the lysates were immunoprecipitated with either control IgG or anti-Myc antibody. The immunoprecipitants were subjected to qPCR analysis using a primer pair to detect the core region of HBV. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).

    Techniques: Expressing, Plasmid Preparation, Transfection, Cotransfection, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction

    FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 protein facilitates cccDNA formation in vitro . (A) Schematic presentation of in vitro cccDNA formation assay. Purified NC-DNA (10 8 copies) was incubated with recombinant FEN1, Bst DNA polymerase, and Taq DNA ligase. Following incubation, the DNA was purified and analyzed (B–F). Regions for qPCR amplification (E and F) were indicated as p. The 5.4-kb PstI fragment in HBV plasmid (Control) has a partial HBV sequence but does not have core and intact P genes. (B) cccDNA-selective qPCR. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; *** P

    Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).

    Techniques: In Vitro, Tube Formation Assay, Purification, Incubation, Recombinant, Real-time Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing

    FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Journal: PLoS Pathogens

    Article Title: Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus

    doi: 10.1371/journal.ppat.1007124

    Figure Lengend Snippet: FEN1 siRNA knockdown and CRISPR/Cas9-mediated gene editing reduce cccDNA production. (A–D) Hep38.7-Tet cells were transfected with FEN1 -specific siRNA (siFEN1 #1 or #2) or control (siCtrl), and cultured without tetracycline. Four days after transfection, FEN1 mRNA/protein and HBV DNA were analyzed. (A) FEN1 mRNA quantified by RT-qPCR (normalized by HPRT) (upper panel) and Western blotting of FEN1 protein (lower panel). GAPDH expression is shown as a loading control. FEN1 protein expression gives rise to two bands in our study, which may be due to post-translational modification. (B–C) Levels of HBV DNA in cytoplasmic nucleocapsid (NC) (B) and cccDNA (C). (D) Efficiency of cccDNA formation (cccDNA levels normalized by cytoplasmic NC-DNA) was calculated. Each result represents the mean ± SEM of three independent experiments. Asterisks indicate statistically significant differences; * P

    Article Snippet: NC-DNA (108 copies) was incubated with 32 units (U) of Thermostable FEN1 in ThermoPol Buffer (New England Biolabs) at 65°C for 10 min, followed by incubation with 8 U of Bst DNA polymerase, 40 U of Taq DNA ligase, 100 μM dNTPs, and NAD+ (all from New England Biolabs).

    Techniques: CRISPR, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Expressing, Modification