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kap1 ps824  (Bethyl)


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    Bethyl kap1 ps824
    Kap1 Ps824, supplied by Bethyl, used in various techniques. Bioz Stars score: 94/100, based on 289 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/kap1 ps824/product/Bethyl
    Average 94 stars, based on 289 article reviews
    kap1 ps824 - by Bioz Stars, 2026-02
    94/100 stars

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    ( A ) DNA double-strand break repair activity in PNKP −/− C1 cells measured by western blotting. DNA double-strand breaks were analyzed by <t>pS824-KAP1</t> and pS139-H2AX (γ-H2AX) antibodies after indicated time points recovered from ionizing-radiation (IR) 5 Gy exposure. KAP1 and PCNA antibodies were used as loading control. p53 antibody was used as DNA damage response control. ( B ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA double-strand breaks, was measured by colony formation assay. Cells were treated by IR exposure at indicated dose. ( C–E ) Schematic, representative images and measurement of DNA single-strand breaks induced by hydrogen peroxide (H 2 O 2 ) in U2OS WT and PNKP −/− cells. Single-strand breaks were analyzed by a native BrdU incorporation method. ( F ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA single-strand breaks, was measured by colony formation assay. Cells were treated by H 2 O 2 at indicated dose. ( G ) Cellular sensitivity of PNKP −/− cells to the DNA replication stress was measured by colony formation assay. Cells were treated by hydroxyurea (HU) at indicated dose. ( H ) Flowcytometric analysis for cell cycle distribution of U2OS WT and PNKP −/− C1 and C2 cells. The cells were stained with propidium iodide (PI), and synthesized DNA was labeled by EdU. Percentage of each cell cycle is shown in vertical axis, cell types are shown in horizontal axis. Statistical analysis of S phase population between WT and PNKP −/− C1 and C2 cells was shown above each bar graph. In all panels, scale bar indicates 10 μm. Statistical significance was indicated as not significant (ns), *: 0.01< p ≦ 0.05 and ****: 0.0005 < p ≦ 0.001. Figure 1—figure supplement 2—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 1—figure supplement 2—source data 2. Original membranes corresponding to .
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    ( A ) DNA double-strand break repair activity in PNKP −/− C1 cells measured by western blotting. DNA double-strand breaks were analyzed by <t>pS824-KAP1</t> and pS139-H2AX (γ-H2AX) antibodies after indicated time points recovered from ionizing-radiation (IR) 5 Gy exposure. KAP1 and PCNA antibodies were used as loading control. p53 antibody was used as DNA damage response control. ( B ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA double-strand breaks, was measured by colony formation assay. Cells were treated by IR exposure at indicated dose. ( C–E ) Schematic, representative images and measurement of DNA single-strand breaks induced by hydrogen peroxide (H 2 O 2 ) in U2OS WT and PNKP −/− cells. Single-strand breaks were analyzed by a native BrdU incorporation method. ( F ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA single-strand breaks, was measured by colony formation assay. Cells were treated by H 2 O 2 at indicated dose. ( G ) Cellular sensitivity of PNKP −/− cells to the DNA replication stress was measured by colony formation assay. Cells were treated by hydroxyurea (HU) at indicated dose. ( H ) Flowcytometric analysis for cell cycle distribution of U2OS WT and PNKP −/− C1 and C2 cells. The cells were stained with propidium iodide (PI), and synthesized DNA was labeled by EdU. Percentage of each cell cycle is shown in vertical axis, cell types are shown in horizontal axis. Statistical analysis of S phase population between WT and PNKP −/− C1 and C2 cells was shown above each bar graph. In all panels, scale bar indicates 10 μm. Statistical significance was indicated as not significant (ns), *: 0.01< p ≦ 0.05 and ****: 0.0005 < p ≦ 0.001. Figure 1—figure supplement 2—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 1—figure supplement 2—source data 2. Original membranes corresponding to .
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    ( A ) DNA double-strand break repair activity in PNKP −/− C1 cells measured by western blotting. DNA double-strand breaks were analyzed by <t>pS824-KAP1</t> and pS139-H2AX (γ-H2AX) antibodies after indicated time points recovered from ionizing-radiation (IR) 5 Gy exposure. KAP1 and PCNA antibodies were used as loading control. p53 antibody was used as DNA damage response control. ( B ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA double-strand breaks, was measured by colony formation assay. Cells were treated by IR exposure at indicated dose. ( C–E ) Schematic, representative images and measurement of DNA single-strand breaks induced by hydrogen peroxide (H 2 O 2 ) in U2OS WT and PNKP −/− cells. Single-strand breaks were analyzed by a native BrdU incorporation method. ( F ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA single-strand breaks, was measured by colony formation assay. Cells were treated by H 2 O 2 at indicated dose. ( G ) Cellular sensitivity of PNKP −/− cells to the DNA replication stress was measured by colony formation assay. Cells were treated by hydroxyurea (HU) at indicated dose. ( H ) Flowcytometric analysis for cell cycle distribution of U2OS WT and PNKP −/− C1 and C2 cells. The cells were stained with propidium iodide (PI), and synthesized DNA was labeled by EdU. Percentage of each cell cycle is shown in vertical axis, cell types are shown in horizontal axis. Statistical analysis of S phase population between WT and PNKP −/− C1 and C2 cells was shown above each bar graph. In all panels, scale bar indicates 10 μm. Statistical significance was indicated as not significant (ns), *: 0.01< p ≦ 0.05 and ****: 0.0005 < p ≦ 0.001. Figure 1—figure supplement 2—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 1—figure supplement 2—source data 2. Original membranes corresponding to .
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    ( A ) DNA double-strand break repair activity in PNKP −/− C1 cells measured by western blotting. DNA double-strand breaks were analyzed by pS824-KAP1 and pS139-H2AX (γ-H2AX) antibodies after indicated time points recovered from ionizing-radiation (IR) 5 Gy exposure. KAP1 and PCNA antibodies were used as loading control. p53 antibody was used as DNA damage response control. ( B ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA double-strand breaks, was measured by colony formation assay. Cells were treated by IR exposure at indicated dose. ( C–E ) Schematic, representative images and measurement of DNA single-strand breaks induced by hydrogen peroxide (H 2 O 2 ) in U2OS WT and PNKP −/− cells. Single-strand breaks were analyzed by a native BrdU incorporation method. ( F ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA single-strand breaks, was measured by colony formation assay. Cells were treated by H 2 O 2 at indicated dose. ( G ) Cellular sensitivity of PNKP −/− cells to the DNA replication stress was measured by colony formation assay. Cells were treated by hydroxyurea (HU) at indicated dose. ( H ) Flowcytometric analysis for cell cycle distribution of U2OS WT and PNKP −/− C1 and C2 cells. The cells were stained with propidium iodide (PI), and synthesized DNA was labeled by EdU. Percentage of each cell cycle is shown in vertical axis, cell types are shown in horizontal axis. Statistical analysis of S phase population between WT and PNKP −/− C1 and C2 cells was shown above each bar graph. In all panels, scale bar indicates 10 μm. Statistical significance was indicated as not significant (ns), *: 0.01< p ≦ 0.05 and ****: 0.0005 < p ≦ 0.001. Figure 1—figure supplement 2—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 1—figure supplement 2—source data 2. Original membranes corresponding to .

    Journal: eLife

    Article Title: CDK-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki fragments and genome stability

    doi: 10.7554/eLife.99217

    Figure Lengend Snippet: ( A ) DNA double-strand break repair activity in PNKP −/− C1 cells measured by western blotting. DNA double-strand breaks were analyzed by pS824-KAP1 and pS139-H2AX (γ-H2AX) antibodies after indicated time points recovered from ionizing-radiation (IR) 5 Gy exposure. KAP1 and PCNA antibodies were used as loading control. p53 antibody was used as DNA damage response control. ( B ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA double-strand breaks, was measured by colony formation assay. Cells were treated by IR exposure at indicated dose. ( C–E ) Schematic, representative images and measurement of DNA single-strand breaks induced by hydrogen peroxide (H 2 O 2 ) in U2OS WT and PNKP −/− cells. Single-strand breaks were analyzed by a native BrdU incorporation method. ( F ) Cellular sensitivity of PNKP −/− cells to the DNA damages, especially DNA single-strand breaks, was measured by colony formation assay. Cells were treated by H 2 O 2 at indicated dose. ( G ) Cellular sensitivity of PNKP −/− cells to the DNA replication stress was measured by colony formation assay. Cells were treated by hydroxyurea (HU) at indicated dose. ( H ) Flowcytometric analysis for cell cycle distribution of U2OS WT and PNKP −/− C1 and C2 cells. The cells were stained with propidium iodide (PI), and synthesized DNA was labeled by EdU. Percentage of each cell cycle is shown in vertical axis, cell types are shown in horizontal axis. Statistical analysis of S phase population between WT and PNKP −/− C1 and C2 cells was shown above each bar graph. In all panels, scale bar indicates 10 μm. Statistical significance was indicated as not significant (ns), *: 0.01< p ≦ 0.05 and ****: 0.0005 < p ≦ 0.001. Figure 1—figure supplement 2—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 1—figure supplement 2—source data 2. Original membranes corresponding to .

    Article Snippet: For primary antibody reactions, the following primary antibodies were used for 1–4 hr at room temperature: PNKP (rabbit, 1:1000, Novus, cat# NBP1-87257), PNKP (rabbit, 1:1000, Abcam, cat# ab181107), pT118-PNKP (rabbit, 1:1000, generated in this paper), pS114-PNKP (rabbit, 1:1000, generated in this paper), XRCC1 (mouse, 1:1000, Invitrogen, Thermo Fisher Scientific, cat# MA5-13412), XRCC4 (rabbit, 1:1000, generated in our laboratory; ), PCNA (rabbit, 1:500, Santa Cruz Biotechnology, cat# sc-7907), KAP1 (rabbit, 1:1000, abcam, cat# ab10483), pS824-KAP1 (rabbit, 1:1000, BETHYL, cat# A300-767A-2), Cyclin-A2 (mouse, 1:1000, Cell signaling, cat# BF683), Cyclin-E1 (rabbit, 1:1000, Sigma-Aldrich, cat# C4976), GFP (mouse, 1:3000, Nacali Tesque Inc, cat# GF200), ATM (mouse, 1:2000, Sigma-Aldrich, cat# A1106), DNA-PKcs (rabbit, 1:2000, abcam, cat# Y393), p53 (mouse, 1:5000, Santa Cruz Biotechnology, cat# sc-126), RPA2 (mouse, 1:3000, abcam, cat# ab2175), GAPDH (mouse, 1:10,000, EMD Millipore, cat# MAB374), FLAG-M2 (mouse, 1:1000, Sigma-Aldrich, cat# A8592), RFP (rabbit, 1:1000, MBL, PM005), and FEN1 (mouse, 1:500, Santa Cruz, sc-28355).

    Techniques: Activity Assay, Western Blot, Control, Colony Assay, BrdU Incorporation Assay, Staining, Synthesized, Labeling

    ( A ) Protein expression analysis of U2OS WT and PNKP −/− C1 cells transiently expressing GFP or indicated PNKP deletion mutants. Protein expression was detected by western blotting. GFP antibody was used for confirming the expression of exogenous PNKP deletion mutants. PNKP antibody (Novus: NBP1-87257) was used for comparing expression levels of endogenous and exogenous PNKP. KAP1 antibody was used as a loading control. ‘*’ indicates endogenous PNKP. ( B, C ) Protein expression analysis of U2OS WT and PNKP −/− C1 cells transiently expressing GFP or indicated PNKP point mutants. Protein expression was detected by western blotting ( B ) and immunofluorescence ( C ). ( B ) GFP antibody was used for confirming the expression of exogenous PNKP point mutants. PNKP antibody was used for comparing expression levels of endogenous and exogenous PNKP. KAP1 antibody was used as a loading control. ‘*’ indicates endogenous PNKP. ( C ) GFP fluorescence was observed by fluorescent microscope. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was used as nucleus staining. In all panels, scale bar indicates 10 μm. Figure 3—figure supplement 1—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 3—figure supplement 1—source data 2. Original membranes corresponding to . Figure 3—figure supplement 1—source data 3. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 3—figure supplement 1—source data 4. Original membranes corresponding to .

    Journal: eLife

    Article Title: CDK-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki fragments and genome stability

    doi: 10.7554/eLife.99217

    Figure Lengend Snippet: ( A ) Protein expression analysis of U2OS WT and PNKP −/− C1 cells transiently expressing GFP or indicated PNKP deletion mutants. Protein expression was detected by western blotting. GFP antibody was used for confirming the expression of exogenous PNKP deletion mutants. PNKP antibody (Novus: NBP1-87257) was used for comparing expression levels of endogenous and exogenous PNKP. KAP1 antibody was used as a loading control. ‘*’ indicates endogenous PNKP. ( B, C ) Protein expression analysis of U2OS WT and PNKP −/− C1 cells transiently expressing GFP or indicated PNKP point mutants. Protein expression was detected by western blotting ( B ) and immunofluorescence ( C ). ( B ) GFP antibody was used for confirming the expression of exogenous PNKP point mutants. PNKP antibody was used for comparing expression levels of endogenous and exogenous PNKP. KAP1 antibody was used as a loading control. ‘*’ indicates endogenous PNKP. ( C ) GFP fluorescence was observed by fluorescent microscope. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was used as nucleus staining. In all panels, scale bar indicates 10 μm. Figure 3—figure supplement 1—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 3—figure supplement 1—source data 2. Original membranes corresponding to . Figure 3—figure supplement 1—source data 3. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 3—figure supplement 1—source data 4. Original membranes corresponding to .

    Article Snippet: For primary antibody reactions, the following primary antibodies were used for 1–4 hr at room temperature: PNKP (rabbit, 1:1000, Novus, cat# NBP1-87257), PNKP (rabbit, 1:1000, Abcam, cat# ab181107), pT118-PNKP (rabbit, 1:1000, generated in this paper), pS114-PNKP (rabbit, 1:1000, generated in this paper), XRCC1 (mouse, 1:1000, Invitrogen, Thermo Fisher Scientific, cat# MA5-13412), XRCC4 (rabbit, 1:1000, generated in our laboratory; ), PCNA (rabbit, 1:500, Santa Cruz Biotechnology, cat# sc-7907), KAP1 (rabbit, 1:1000, abcam, cat# ab10483), pS824-KAP1 (rabbit, 1:1000, BETHYL, cat# A300-767A-2), Cyclin-A2 (mouse, 1:1000, Cell signaling, cat# BF683), Cyclin-E1 (rabbit, 1:1000, Sigma-Aldrich, cat# C4976), GFP (mouse, 1:3000, Nacali Tesque Inc, cat# GF200), ATM (mouse, 1:2000, Sigma-Aldrich, cat# A1106), DNA-PKcs (rabbit, 1:2000, abcam, cat# Y393), p53 (mouse, 1:5000, Santa Cruz Biotechnology, cat# sc-126), RPA2 (mouse, 1:3000, abcam, cat# ab2175), GAPDH (mouse, 1:10,000, EMD Millipore, cat# MAB374), FLAG-M2 (mouse, 1:1000, Sigma-Aldrich, cat# A8592), RFP (rabbit, 1:1000, MBL, PM005), and FEN1 (mouse, 1:500, Santa Cruz, sc-28355).

    Techniques: Expressing, Western Blot, Control, Immunofluorescence, Fluorescence, Microscopy, Staining

    ( A ) Scheme and protein expression levels of GFP-PNKP-expressing HCT116 cells after release from double thymidine block. After released, cells were collected at indicated time points, and used for western blotting. pT118 PNKP antibody was generated for this study. Cyclin A2 and E1 antibodies were used for cell cycle markers. GAPDH antibody was used as a loading control. ( B ) In vitro analysis of PNKP phosphorylation on T118 by CDK/Cyclin complex. Purified His-PNKP and each CDK/Cyclin complex were incubated with reaction mixture and detected with western blotting by pT118-PNKP, His, GST, and Cyclin A2 antibodies. ( C ) HCT116 cells were lysed at 5 and 3 days after transfection with GFP-PNKP and co-transfection with mCherry2-Cyclin A2, and 3XFLAG-CDK1 or CDK2, respectively, and applied for western blotting. pT118 PNKP level was analyzed by pT118 PNKP-specific antibody. PNKP expression was analyzed with PNKP or GFP antibodies. Cyclin A2 expression was analyzed with RFP antibody. KAP1 antibody was used as loading control. ( D ) Analysis of isolated proteins from nascent DNA using isolation of proteins on nascent DNA (iPOND) technique. Proteins bound to EdU-labeled DNA in GFP-PNKP-expressing HEK293 cells were isolated using click reaction with biotin-azide followed by streptavidin-pulldowns and detected by western blotting. 0.1% of lysate used in Streptavidin-pulldowns represented as 0.1% input. ( E ) Western blotting to show interactions of GFP-PNKP WT or T118A with nascent DNA using the iPOND technique. PCNA was used as a loading control. ( F ) Western blotting to show interaction of PNKP with nascent DNA in XRCC1-depleted cells using the iPOND technique. PCNA was used as a loading control. Figure 4—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 2. Original membranes corresponding to . Figure 4—source data 3. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 4. Original membranes corresponding to . Figure 4—source data 5. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 6. Original membranes corresponding to . Figure 4—source data 7. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 8. Original membranes corresponding to . Figure 4—source data 9. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 10. Original membranes corresponding to . Figure 4—source data 11. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 12. Original membranes corresponding to .

    Journal: eLife

    Article Title: CDK-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki fragments and genome stability

    doi: 10.7554/eLife.99217

    Figure Lengend Snippet: ( A ) Scheme and protein expression levels of GFP-PNKP-expressing HCT116 cells after release from double thymidine block. After released, cells were collected at indicated time points, and used for western blotting. pT118 PNKP antibody was generated for this study. Cyclin A2 and E1 antibodies were used for cell cycle markers. GAPDH antibody was used as a loading control. ( B ) In vitro analysis of PNKP phosphorylation on T118 by CDK/Cyclin complex. Purified His-PNKP and each CDK/Cyclin complex were incubated with reaction mixture and detected with western blotting by pT118-PNKP, His, GST, and Cyclin A2 antibodies. ( C ) HCT116 cells were lysed at 5 and 3 days after transfection with GFP-PNKP and co-transfection with mCherry2-Cyclin A2, and 3XFLAG-CDK1 or CDK2, respectively, and applied for western blotting. pT118 PNKP level was analyzed by pT118 PNKP-specific antibody. PNKP expression was analyzed with PNKP or GFP antibodies. Cyclin A2 expression was analyzed with RFP antibody. KAP1 antibody was used as loading control. ( D ) Analysis of isolated proteins from nascent DNA using isolation of proteins on nascent DNA (iPOND) technique. Proteins bound to EdU-labeled DNA in GFP-PNKP-expressing HEK293 cells were isolated using click reaction with biotin-azide followed by streptavidin-pulldowns and detected by western blotting. 0.1% of lysate used in Streptavidin-pulldowns represented as 0.1% input. ( E ) Western blotting to show interactions of GFP-PNKP WT or T118A with nascent DNA using the iPOND technique. PCNA was used as a loading control. ( F ) Western blotting to show interaction of PNKP with nascent DNA in XRCC1-depleted cells using the iPOND technique. PCNA was used as a loading control. Figure 4—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 2. Original membranes corresponding to . Figure 4—source data 3. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 4. Original membranes corresponding to . Figure 4—source data 5. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 6. Original membranes corresponding to . Figure 4—source data 7. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 8. Original membranes corresponding to . Figure 4—source data 9. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 10. Original membranes corresponding to . Figure 4—source data 11. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 4—source data 12. Original membranes corresponding to .

    Article Snippet: For primary antibody reactions, the following primary antibodies were used for 1–4 hr at room temperature: PNKP (rabbit, 1:1000, Novus, cat# NBP1-87257), PNKP (rabbit, 1:1000, Abcam, cat# ab181107), pT118-PNKP (rabbit, 1:1000, generated in this paper), pS114-PNKP (rabbit, 1:1000, generated in this paper), XRCC1 (mouse, 1:1000, Invitrogen, Thermo Fisher Scientific, cat# MA5-13412), XRCC4 (rabbit, 1:1000, generated in our laboratory; ), PCNA (rabbit, 1:500, Santa Cruz Biotechnology, cat# sc-7907), KAP1 (rabbit, 1:1000, abcam, cat# ab10483), pS824-KAP1 (rabbit, 1:1000, BETHYL, cat# A300-767A-2), Cyclin-A2 (mouse, 1:1000, Cell signaling, cat# BF683), Cyclin-E1 (rabbit, 1:1000, Sigma-Aldrich, cat# C4976), GFP (mouse, 1:3000, Nacali Tesque Inc, cat# GF200), ATM (mouse, 1:2000, Sigma-Aldrich, cat# A1106), DNA-PKcs (rabbit, 1:2000, abcam, cat# Y393), p53 (mouse, 1:5000, Santa Cruz Biotechnology, cat# sc-126), RPA2 (mouse, 1:3000, abcam, cat# ab2175), GAPDH (mouse, 1:10,000, EMD Millipore, cat# MAB374), FLAG-M2 (mouse, 1:1000, Sigma-Aldrich, cat# A8592), RFP (rabbit, 1:1000, MBL, PM005), and FEN1 (mouse, 1:500, Santa Cruz, sc-28355).

    Techniques: Expressing, Blocking Assay, Western Blot, Generated, Control, In Vitro, Phospho-proteomics, Purification, Incubation, Transfection, Cotransfection, Isolation, Labeling

    ( A ) Protein expression analysis of U2OS WT and PNKP −/− C1 cells transiently expressing GFP or indicated PNKP mutants. Protein expression was detected by western blotting. GFP antibody was used for confirming the expression of exogenous PNKP mutants. PNKP antibody was used for comparing expression levels of endogenous and exogenous PNKP. KAP1 antibody was used as a loading control. ‘*’ indicates endogenous PNKP. ( B ) Quantified results of the experiments for measurement of ADP-ribose intensity in PNKP −/− C1 cells expressing PNKP WT and PNKP T118D mutant under DMSO and FEN1i treatment. Error bars represent SEM. ( C ) Representative images of immunofluorescence using PAN ADP-ribose-binding reagents at 30 min after 2 Gy ionizing-radiation (IR) exposure in U2OS WT and PNKP −/− cells transiently expressing GFP or indicated PNKP mutants. PARGi was added 30 min prior to IR exposure. Images detected by ADP-ribose, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and DIC were shown. Scale bar indicates as 10 μm. ( D ) Quantified result of C . Relative ADP-ribose intensity is shown in vertical axis and cell types were shown in horizontal axis. At least 100 cells were analyzed for the quantification. ( E ) Measurement of ADP-ribose intensity in U2OS WT or PNKP −/− C1 cells expressing GFP, PNKP WT, and PNKP T118A mutant under H 2 O 2 treatment in EdU- and EdU-positive cells. Error bars represent SEM. ( F ) Measurement of DNA double-strand break (DSB) repair ability of indicated cells after 2Gy IR exposure. Cells were harvested at 30 min, 6 hr, and 24 hr after IR exposure. Percentage of γH2AX-positive cells is shown in vertical axis and conditions were shown in horizontal axis. NT indicates non-treatment. At least 100 cells were analyzed for the quantification. Statistical significance was indicated as not significant (ns) and ****: 0.0005 < p ≦ 0.001. ( G, H ) Measurement of the formations of micronuclei and chromosome bridges in U2OS WT and PNKP −/− C1 cells expressing GFP, PNKP WT, and PNKP T118A mutant exposed to 5 Gy IR or 2 mM hydroxyurea (HU). DNA were stained by DAPI at 24 hr after each treatment. Cells with micronucleus ( G ) and chromosome bridge ( H ) were counted and graphed. At least 200 cells were counted. Error bars represent SEM. In all panels, statistical significance was indicated as not significant (ns), *: 0.01< p ≦ 0.05, **: 0.005< p ≦ 0.01, ***: 0.001 < p ≦ 0.005 and ****: 0.0005 < p ≦ 0.001. Figure 5—figure supplement 1—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 5—figure supplement 1—source data 2. Original membranes corresponding to .

    Journal: eLife

    Article Title: CDK-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki fragments and genome stability

    doi: 10.7554/eLife.99217

    Figure Lengend Snippet: ( A ) Protein expression analysis of U2OS WT and PNKP −/− C1 cells transiently expressing GFP or indicated PNKP mutants. Protein expression was detected by western blotting. GFP antibody was used for confirming the expression of exogenous PNKP mutants. PNKP antibody was used for comparing expression levels of endogenous and exogenous PNKP. KAP1 antibody was used as a loading control. ‘*’ indicates endogenous PNKP. ( B ) Quantified results of the experiments for measurement of ADP-ribose intensity in PNKP −/− C1 cells expressing PNKP WT and PNKP T118D mutant under DMSO and FEN1i treatment. Error bars represent SEM. ( C ) Representative images of immunofluorescence using PAN ADP-ribose-binding reagents at 30 min after 2 Gy ionizing-radiation (IR) exposure in U2OS WT and PNKP −/− cells transiently expressing GFP or indicated PNKP mutants. PARGi was added 30 min prior to IR exposure. Images detected by ADP-ribose, 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) and DIC were shown. Scale bar indicates as 10 μm. ( D ) Quantified result of C . Relative ADP-ribose intensity is shown in vertical axis and cell types were shown in horizontal axis. At least 100 cells were analyzed for the quantification. ( E ) Measurement of ADP-ribose intensity in U2OS WT or PNKP −/− C1 cells expressing GFP, PNKP WT, and PNKP T118A mutant under H 2 O 2 treatment in EdU- and EdU-positive cells. Error bars represent SEM. ( F ) Measurement of DNA double-strand break (DSB) repair ability of indicated cells after 2Gy IR exposure. Cells were harvested at 30 min, 6 hr, and 24 hr after IR exposure. Percentage of γH2AX-positive cells is shown in vertical axis and conditions were shown in horizontal axis. NT indicates non-treatment. At least 100 cells were analyzed for the quantification. Statistical significance was indicated as not significant (ns) and ****: 0.0005 < p ≦ 0.001. ( G, H ) Measurement of the formations of micronuclei and chromosome bridges in U2OS WT and PNKP −/− C1 cells expressing GFP, PNKP WT, and PNKP T118A mutant exposed to 5 Gy IR or 2 mM hydroxyurea (HU). DNA were stained by DAPI at 24 hr after each treatment. Cells with micronucleus ( G ) and chromosome bridge ( H ) were counted and graphed. At least 200 cells were counted. Error bars represent SEM. In all panels, statistical significance was indicated as not significant (ns), *: 0.01< p ≦ 0.05, **: 0.005< p ≦ 0.01, ***: 0.001 < p ≦ 0.005 and ****: 0.0005 < p ≦ 0.001. Figure 5—figure supplement 1—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 5—figure supplement 1—source data 2. Original membranes corresponding to .

    Article Snippet: For primary antibody reactions, the following primary antibodies were used for 1–4 hr at room temperature: PNKP (rabbit, 1:1000, Novus, cat# NBP1-87257), PNKP (rabbit, 1:1000, Abcam, cat# ab181107), pT118-PNKP (rabbit, 1:1000, generated in this paper), pS114-PNKP (rabbit, 1:1000, generated in this paper), XRCC1 (mouse, 1:1000, Invitrogen, Thermo Fisher Scientific, cat# MA5-13412), XRCC4 (rabbit, 1:1000, generated in our laboratory; ), PCNA (rabbit, 1:500, Santa Cruz Biotechnology, cat# sc-7907), KAP1 (rabbit, 1:1000, abcam, cat# ab10483), pS824-KAP1 (rabbit, 1:1000, BETHYL, cat# A300-767A-2), Cyclin-A2 (mouse, 1:1000, Cell signaling, cat# BF683), Cyclin-E1 (rabbit, 1:1000, Sigma-Aldrich, cat# C4976), GFP (mouse, 1:3000, Nacali Tesque Inc, cat# GF200), ATM (mouse, 1:2000, Sigma-Aldrich, cat# A1106), DNA-PKcs (rabbit, 1:2000, abcam, cat# Y393), p53 (mouse, 1:5000, Santa Cruz Biotechnology, cat# sc-126), RPA2 (mouse, 1:3000, abcam, cat# ab2175), GAPDH (mouse, 1:10,000, EMD Millipore, cat# MAB374), FLAG-M2 (mouse, 1:1000, Sigma-Aldrich, cat# A8592), RFP (rabbit, 1:1000, MBL, PM005), and FEN1 (mouse, 1:500, Santa Cruz, sc-28355).

    Techniques: Expressing, Western Blot, Control, Mutagenesis, Immunofluorescence, Binding Assay, Staining

    ( A ) Protein expression analysis for PNKP phosphatase-dead (D171A) and kinase-dead (K378A) mutants in U2OS WT and PNKP −/− C1 cells expressing GFP, PNKP WT, D171A, and K378A mutants confirmed by western blotting. KAP1 antibody was used as a loading control. ( B ) Indicated number of cell extracts harvested from U2OS WT and PNKP −/− C1 cells were incubated with TAMRA or 6-FAM-labeled oligonucleotide duplex harboring a single-strand break (SSB) GAP structure. Arrows indicate the positions of the TAMRA-labeled phosphatase substrates (top) and 6-FAM-labeled kinase substrates (bottom). ( C ) PNKP phosphatase and kinase activity biochemical analysis. Indicated cell extracts harvested from PNKP −/− C1 cells expressing PNKP WT, T118A, D171A (phosphatase-dead), and K378A (kinase-dead) were incubated with TAMRA or 6-FAM-labeled oligonucleotide duplex harboring a single-strand DNA (ssDNA) gap structure. Arrows indicate the positions of the TAMRA-labeled phosphatase substrates (left) and 6-FAM-labeled kinase substrates (right). Band intensity of 18-nt 3′-OH (left) and 21-nt 5′-P (right) were analyzed using ImageJ software and indicated. ( D ) Measurement of the gapped DNA-binding ability of PNKP WT, T118A, and T118D mutants. Nuclear extracts were harvested from U2OS PNKP −/− C1 cells at 2 days after transfection with GFP-PNKP WT, T118A, or T118D mutants. GFP antibody was used to detect GFP-PNKPs bound to the gapped double-stranded DNA. Error bars represent SEM. Statistical significance was indicated as not significant (ns) and ****: 0.0005 < p ≦ 0.001. Figure 6—figure supplement 1—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 6—figure supplement 1—source data 2. Original membranes corresponding to . Figure 6—figure supplement 1—source data 3. Original gels corresponding to . Indicated number of cell extracts harvested from U2OS WT and PNKP −/− C1 cells were incubated with TAMRA or 6-FAM-labeled oligonucleotide duplex harboring a single-strand break (SSB) GAP structure. Arrows indicate the positions of the TAMRA-labeled phosphatase substrates (top: 1/2) and 6-FAM-labeled kinase substrates (bottom: 2/2). Figure 6—figure supplement 1—source data 4. Original gels corresponding to . Figure 6—figure supplement 1—source data 5. Original gels corresponding to . Regions surrounded with red dashed line represent cropped areas. Figure 6—figure supplement 1—source data 6. Original gels corresponding to .

    Journal: eLife

    Article Title: CDK-mediated phosphorylation of PNKP is required for end-processing of single-strand DNA gaps on Okazaki fragments and genome stability

    doi: 10.7554/eLife.99217

    Figure Lengend Snippet: ( A ) Protein expression analysis for PNKP phosphatase-dead (D171A) and kinase-dead (K378A) mutants in U2OS WT and PNKP −/− C1 cells expressing GFP, PNKP WT, D171A, and K378A mutants confirmed by western blotting. KAP1 antibody was used as a loading control. ( B ) Indicated number of cell extracts harvested from U2OS WT and PNKP −/− C1 cells were incubated with TAMRA or 6-FAM-labeled oligonucleotide duplex harboring a single-strand break (SSB) GAP structure. Arrows indicate the positions of the TAMRA-labeled phosphatase substrates (top) and 6-FAM-labeled kinase substrates (bottom). ( C ) PNKP phosphatase and kinase activity biochemical analysis. Indicated cell extracts harvested from PNKP −/− C1 cells expressing PNKP WT, T118A, D171A (phosphatase-dead), and K378A (kinase-dead) were incubated with TAMRA or 6-FAM-labeled oligonucleotide duplex harboring a single-strand DNA (ssDNA) gap structure. Arrows indicate the positions of the TAMRA-labeled phosphatase substrates (left) and 6-FAM-labeled kinase substrates (right). Band intensity of 18-nt 3′-OH (left) and 21-nt 5′-P (right) were analyzed using ImageJ software and indicated. ( D ) Measurement of the gapped DNA-binding ability of PNKP WT, T118A, and T118D mutants. Nuclear extracts were harvested from U2OS PNKP −/− C1 cells at 2 days after transfection with GFP-PNKP WT, T118A, or T118D mutants. GFP antibody was used to detect GFP-PNKPs bound to the gapped double-stranded DNA. Error bars represent SEM. Statistical significance was indicated as not significant (ns) and ****: 0.0005 < p ≦ 0.001. Figure 6—figure supplement 1—source data 1. Original membranes corresponding to . Regions surrounded with red dashed line represent cropped areas, respectively. Annotations represent employed antibodies, respectively. Figure 6—figure supplement 1—source data 2. Original membranes corresponding to . Figure 6—figure supplement 1—source data 3. Original gels corresponding to . Indicated number of cell extracts harvested from U2OS WT and PNKP −/− C1 cells were incubated with TAMRA or 6-FAM-labeled oligonucleotide duplex harboring a single-strand break (SSB) GAP structure. Arrows indicate the positions of the TAMRA-labeled phosphatase substrates (top: 1/2) and 6-FAM-labeled kinase substrates (bottom: 2/2). Figure 6—figure supplement 1—source data 4. Original gels corresponding to . Figure 6—figure supplement 1—source data 5. Original gels corresponding to . Regions surrounded with red dashed line represent cropped areas. Figure 6—figure supplement 1—source data 6. Original gels corresponding to .

    Article Snippet: For primary antibody reactions, the following primary antibodies were used for 1–4 hr at room temperature: PNKP (rabbit, 1:1000, Novus, cat# NBP1-87257), PNKP (rabbit, 1:1000, Abcam, cat# ab181107), pT118-PNKP (rabbit, 1:1000, generated in this paper), pS114-PNKP (rabbit, 1:1000, generated in this paper), XRCC1 (mouse, 1:1000, Invitrogen, Thermo Fisher Scientific, cat# MA5-13412), XRCC4 (rabbit, 1:1000, generated in our laboratory; ), PCNA (rabbit, 1:500, Santa Cruz Biotechnology, cat# sc-7907), KAP1 (rabbit, 1:1000, abcam, cat# ab10483), pS824-KAP1 (rabbit, 1:1000, BETHYL, cat# A300-767A-2), Cyclin-A2 (mouse, 1:1000, Cell signaling, cat# BF683), Cyclin-E1 (rabbit, 1:1000, Sigma-Aldrich, cat# C4976), GFP (mouse, 1:3000, Nacali Tesque Inc, cat# GF200), ATM (mouse, 1:2000, Sigma-Aldrich, cat# A1106), DNA-PKcs (rabbit, 1:2000, abcam, cat# Y393), p53 (mouse, 1:5000, Santa Cruz Biotechnology, cat# sc-126), RPA2 (mouse, 1:3000, abcam, cat# ab2175), GAPDH (mouse, 1:10,000, EMD Millipore, cat# MAB374), FLAG-M2 (mouse, 1:1000, Sigma-Aldrich, cat# A8592), RFP (rabbit, 1:1000, MBL, PM005), and FEN1 (mouse, 1:500, Santa Cruz, sc-28355).

    Techniques: Expressing, Western Blot, Control, Incubation, Labeling, Activity Assay, Software, Binding Assay, Transfection