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    Cell Signaling Technology Inc etdrs letters 60 5 11 0 60 8 10 6 62 mean cst
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    Cell Signaling Technology Inc ddb2
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    Cell Signaling Technology Inc ddb2
    ERα mediates chemotherapy-induced <t>DDB2</t> gene transcription (A and B) Basal mRNA (A) and protein (B) levels of DDB2 were higher in ER-positive than ER-negative breast cancer cell lines, regardless of their TP53 genetic status. (C) Silencing of ERα by two independent shRNAs suppressed DDB2 protein and mRNA expression. Reduction of both S118-phosphorylated and total ERα revealed the inhibition of ERα activity. (D) ER response elements (EREs) and p53 RE (p53RE) motifs on the DDB2 promoter and the luciferase reporter constructs driven by different lengths of the DDB2 promoter were illustrated (vermilion, red, and pink: EREs; orange: p53RE). (E) Treatment with carboplatin (50 μM) increased the luciferase activity driven by the DDB2 promoter containing both p53RE and EREs in mutp53-expressing T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity. (F) Silencing of ERα decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells, and firefly luciferase activity was normalized with β-gal activity. Data in (A), (C), (E), and (F) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
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    1) Product Images from "ERα determines the chemo-resistant function of mutant p53 involving the switch between lincRNA-p21 and DDB2 expressions"

    Article Title: ERα determines the chemo-resistant function of mutant p53 involving the switch between lincRNA-p21 and DDB2 expressions

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2021.07.022

    ERα mediates chemotherapy-induced DDB2 gene transcription (A and B) Basal mRNA (A) and protein (B) levels of DDB2 were higher in ER-positive than ER-negative breast cancer cell lines, regardless of their TP53 genetic status. (C) Silencing of ERα by two independent shRNAs suppressed DDB2 protein and mRNA expression. Reduction of both S118-phosphorylated and total ERα revealed the inhibition of ERα activity. (D) ER response elements (EREs) and p53 RE (p53RE) motifs on the DDB2 promoter and the luciferase reporter constructs driven by different lengths of the DDB2 promoter were illustrated (vermilion, red, and pink: EREs; orange: p53RE). (E) Treatment with carboplatin (50 μM) increased the luciferase activity driven by the DDB2 promoter containing both p53RE and EREs in mutp53-expressing T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity. (F) Silencing of ERα decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells, and firefly luciferase activity was normalized with β-gal activity. Data in (A), (C), (E), and (F) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
    Figure Legend Snippet: ERα mediates chemotherapy-induced DDB2 gene transcription (A and B) Basal mRNA (A) and protein (B) levels of DDB2 were higher in ER-positive than ER-negative breast cancer cell lines, regardless of their TP53 genetic status. (C) Silencing of ERα by two independent shRNAs suppressed DDB2 protein and mRNA expression. Reduction of both S118-phosphorylated and total ERα revealed the inhibition of ERα activity. (D) ER response elements (EREs) and p53 RE (p53RE) motifs on the DDB2 promoter and the luciferase reporter constructs driven by different lengths of the DDB2 promoter were illustrated (vermilion, red, and pink: EREs; orange: p53RE). (E) Treatment with carboplatin (50 μM) increased the luciferase activity driven by the DDB2 promoter containing both p53RE and EREs in mutp53-expressing T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity. (F) Silencing of ERα decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells, and firefly luciferase activity was normalized with β-gal activity. Data in (A), (C), (E), and (F) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.

    Techniques Used: Expressing, Inhibition, Activity Assay, Luciferase, Construct

    ERα cooperates with mutp53 to upregulate DDB2 gene transcription and mediates chemoresistance in ER-positive breast cancer cells (A) Silencing of p53 decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. (B) Deletions of EREs, but not p53RE, reduced carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity in (A) and (B). (C) Carboplatin (50 μM) induced chromatin-binding affinity of both ERα and mutp53 preferentially on ERE#1 of the DDB2 promoter in T-47D cancer cells in the ChIP assay. (D) Silencing of ERα reduced p53 activity, as evidenced by the acetylation at K382 in T-47D and BT-474 cancer cells. (E) Chemotherapy (50 μM carboplatin or 0.5 μM doxorubicin) induced the protein interaction between p53 and ERα in the coimmunoprecipitation (coIP) assay. (F) Ectopic co-expression of ERα and p53 mutants (R280K and R273H) synergistically enhanced DDB2 expression in HEK293T cells. (G) Silencing of DDB2 by two independent shRNAs enhanced carboplatin (50 μM)-induced DNA damage in the comet assay. The tail moment and tail-length index were calculated from images by Comet Assay III analysis. Data in (A−C) and (G) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
    Figure Legend Snippet: ERα cooperates with mutp53 to upregulate DDB2 gene transcription and mediates chemoresistance in ER-positive breast cancer cells (A) Silencing of p53 decreased the carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. (B) Deletions of EREs, but not p53RE, reduced carboplatin (50 μM)-induced DDB2 promoter activity in T-47D cancer cells. Firefly luciferase activity was normalized with β-gal activity in (A) and (B). (C) Carboplatin (50 μM) induced chromatin-binding affinity of both ERα and mutp53 preferentially on ERE#1 of the DDB2 promoter in T-47D cancer cells in the ChIP assay. (D) Silencing of ERα reduced p53 activity, as evidenced by the acetylation at K382 in T-47D and BT-474 cancer cells. (E) Chemotherapy (50 μM carboplatin or 0.5 μM doxorubicin) induced the protein interaction between p53 and ERα in the coimmunoprecipitation (coIP) assay. (F) Ectopic co-expression of ERα and p53 mutants (R280K and R273H) synergistically enhanced DDB2 expression in HEK293T cells. (G) Silencing of DDB2 by two independent shRNAs enhanced carboplatin (50 μM)-induced DNA damage in the comet assay. The tail moment and tail-length index were calculated from images by Comet Assay III analysis. Data in (A−C) and (G) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.

    Techniques Used: Activity Assay, Luciferase, Binding Assay, Co-Immunoprecipitation Assay, Expressing, Single Cell Gel Electrophoresis

    ERα hijacks mutp53 from the G-quadruplex DNA (GQ) structure of the lincRNA-p21 promoter to EREs of the DDB2 promoter (A) Illustration of the predicted non-B structure and p53RE on the lincRNA-p21 promoter and two luciferase-reporter constructs driven by lincRNA-p21 promoters containing different motifs. (B and C) GQ motif is required for mutp53-increased lincRNA-p21 promoter activity, and firefly luciferase activity was normalized with β-gal activity. (D) The binding efficacy of endogenous mutp53 on different motifs of the lincRNA-p21 promoter in response to chemotherapies (50 μM carboplatin and 0.5 μM doxorubicin) in the ChIP assay. (E and F) Carboplatin (50 μM) and two GQ stabilizers (1 μM NMM and 5 μM auramine) increased the binding of mutp53 to the GQ motif of the 5′-biotinylated lincRNA-p21 promoters in the in vitro pull-down assay. (G) GQ stabilizers increased lincRNA-p21 expression in MDA-MB-231 cancer cells. (H) Silencing of ERα switched the chromatin-binding activity of mutp53 from EREs of the DDB2 promoter (left) to the non-B DNA motifs of the lincRNA-p21 promoter (right) in ER-positive/p53 L194F T-47D cancer cells. (I) The inversed correlation between the ex vivo induction of lincRNA-p21 and DDB2 expression by carboplatin (50 μM) treatments in human primary breast cancer tissues in an ER status-dependent manner. Data in (B−D), (G), and (H) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.
    Figure Legend Snippet: ERα hijacks mutp53 from the G-quadruplex DNA (GQ) structure of the lincRNA-p21 promoter to EREs of the DDB2 promoter (A) Illustration of the predicted non-B structure and p53RE on the lincRNA-p21 promoter and two luciferase-reporter constructs driven by lincRNA-p21 promoters containing different motifs. (B and C) GQ motif is required for mutp53-increased lincRNA-p21 promoter activity, and firefly luciferase activity was normalized with β-gal activity. (D) The binding efficacy of endogenous mutp53 on different motifs of the lincRNA-p21 promoter in response to chemotherapies (50 μM carboplatin and 0.5 μM doxorubicin) in the ChIP assay. (E and F) Carboplatin (50 μM) and two GQ stabilizers (1 μM NMM and 5 μM auramine) increased the binding of mutp53 to the GQ motif of the 5′-biotinylated lincRNA-p21 promoters in the in vitro pull-down assay. (G) GQ stabilizers increased lincRNA-p21 expression in MDA-MB-231 cancer cells. (H) Silencing of ERα switched the chromatin-binding activity of mutp53 from EREs of the DDB2 promoter (left) to the non-B DNA motifs of the lincRNA-p21 promoter (right) in ER-positive/p53 L194F T-47D cancer cells. (I) The inversed correlation between the ex vivo induction of lincRNA-p21 and DDB2 expression by carboplatin (50 μM) treatments in human primary breast cancer tissues in an ER status-dependent manner. Data in (B−D), (G), and (H) were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test.

    Techniques Used: Luciferase, Construct, Activity Assay, Binding Assay, In Vitro, Pull Down Assay, Expressing, Ex Vivo

    lincRNA-p21 reduces the growth and chemoresistance of the ER-positive tumor in vivo (A) The T-47D#Tet-On-LincRNA-p21 stable clone was treated with or without tetracycline (10 μg/mL), and the expressions of lincRNA-p21 and DDB2 were detected by quantitative real-time PCR analysis and western blot assay, respectively. Data were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (B) Illustration of the treatment timeline in the tumor-xenograft mouse model (yellow arrow: the starting point for tetracycline administration [0.2 mg/mL]; red arrow: the points for the intraperitoneal injection with doxorubicin [2.5 mg/kg]). (C and D) The tumor growth rate (C) and size at the end point (D) in four groups of these mice. Data were representative of n = 3 in every group and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (E) The expressions of lincRNA-p21, DDB2, Ki67, and cleaved caspase-3 in the tumor tissues were examined in the in situ hybridization and immunohistochemistry assays.
    Figure Legend Snippet: lincRNA-p21 reduces the growth and chemoresistance of the ER-positive tumor in vivo (A) The T-47D#Tet-On-LincRNA-p21 stable clone was treated with or without tetracycline (10 μg/mL), and the expressions of lincRNA-p21 and DDB2 were detected by quantitative real-time PCR analysis and western blot assay, respectively. Data were representative of three experiments and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (B) Illustration of the treatment timeline in the tumor-xenograft mouse model (yellow arrow: the starting point for tetracycline administration [0.2 mg/mL]; red arrow: the points for the intraperitoneal injection with doxorubicin [2.5 mg/kg]). (C and D) The tumor growth rate (C) and size at the end point (D) in four groups of these mice. Data were representative of n = 3 in every group and were shown as the mean ± SD. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001 versus the control group, Student’s t test. (E) The expressions of lincRNA-p21, DDB2, Ki67, and cleaved caspase-3 in the tumor tissues were examined in the in situ hybridization and immunohistochemistry assays.

    Techniques Used: In Vivo, Stable Transfection, Real-time Polymerase Chain Reaction, Western Blot, Injection, In Situ Hybridization, Immunohistochemistry

    The proposed model of ERα/mutp53 mediated DDB2 and lincRNA-p21 in contributing to chemoresistance The proposed model illustrates how ERα determined chemoresistance of breast cancer by disrupting the balance between mutp53-dependent DDB2 and lincRNA-p21 transcriptions. In the ER-negative cancer cells (left), lincRNA-p21 , transcribed by mutp53 in a G-quadruplex of non-B structure-dependent fashion, mediate apoptosis for chemosensitivity. In the ER-positive cancer cells (right), however, ERα switches mutp53 to preferentially mediate DDB2 transcription via targeting its EREs and thereby reduces lincRNA-p21 expression, conferring chemoresistance (p53∗, mutp53).
    Figure Legend Snippet: The proposed model of ERα/mutp53 mediated DDB2 and lincRNA-p21 in contributing to chemoresistance The proposed model illustrates how ERα determined chemoresistance of breast cancer by disrupting the balance between mutp53-dependent DDB2 and lincRNA-p21 transcriptions. In the ER-negative cancer cells (left), lincRNA-p21 , transcribed by mutp53 in a G-quadruplex of non-B structure-dependent fashion, mediate apoptosis for chemosensitivity. In the ER-positive cancer cells (right), however, ERα switches mutp53 to preferentially mediate DDB2 transcription via targeting its EREs and thereby reduces lincRNA-p21 expression, conferring chemoresistance (p53∗, mutp53).

    Techniques Used: Expressing

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    Cell Signaling Technology Inc sc ddb2
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    Cell Signaling Technology Inc anti ddb2
    USP44 deubiquitinates <t>DDB2</t> following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.
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    1) Product Images from "USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors"

    Article Title: USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2021.663411

    USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.
    Figure Legend Snippet: USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.

    Techniques Used: Western Blot, Transduction, Construct, Immunoprecipitation, FLAG-tag, Purification, Incubation, Mutagenesis

    Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.
    Figure Legend Snippet: Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.

    Techniques Used: Expressing, Transfection, Irradiation, Fluorescence, Imaging, Transduction, Mutagenesis, Binding Assay

    ddb2 ab  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ddb2 ab
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    Cell Signaling Technology Inc anti ddb2
    In response to UV irradiation, SIRT6 interacts with <t>DDB2.</t> ( A ) SIRT6 fails to stimulate NER in XPC-depleted HCA2-hTERT cells. A control vector or vector encoding SIRT6 was cotransfected with UVC-treated pmax-GFP and pDsRed2-N1 into control and XPC-depleted HCA2-hTERT cells. At 72 h post transfection, the cells were harvested for FACS analysis. Error bars represent the s.d. *** P < 0.001, n.s., not significant. (B, C) SIRT6 interacts with DDB2 in vivo . HEK293 cells were transfected with Flag-tagged SIRT6. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the Flag tag, followed by western blot analysis with the indicated antibodies ( B ). HEK293 cells were transfected with HA-tagged DDB2. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the HA tag, followed by western blot analysis ( C ). ( D ) SIRT6 interacts with DDB2 in vitro . Five micrograms of recombinant His-DDB2 and GST or GST-SIRT6 together with 30 μl GST-agarose resin were incubated with GST or GST-SIRT6 in IP buffer for 6 h at 4°C. Western blot analysis was performed with the indicated antibodies. ( E ) A schematic representation of the SIRT6 fragments used in this study is shown. ( F ) A control vector or vectors encoding full-length SIRT6-Flag, SIRT6 ΔC-Flag (1-271 aa), SIRT6 ΔN-Flag (49–355 aa) or the deacetylase core fragment SIRT6 ΔNΔC-Flag (49–271 aa) were cotransfected with DDB2-GFP into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with GFP-Trap (Chromotek), followed by western blot analysis. ( G ) A schematic representation of the DDB2 fragments used in this study is shown. ( H ) Vectors encoding full-length DDB2-GFP, DDB2 fragment 1-GFP (1–100 aa), or DDB2 fragment 2-GFP (101–427 aa) were cotransfected with a SIRT6-Flag-expressing vector into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis. ( I ) SIRT6 fails to stimulate NER in DDB2-depleted HCA2-hTERT cells. HCA2-hTERT cells were transfected with control siRNA or DDB2-specific siRNA twice over a 48-h interval. Afterwards, the HCA2-hTERT cells were transfected with a control vector or a vector expressing SIRT6 and UVC-treated pmax-GFP together with pDsRed2-N1. On day 3 post transfection, the cells were harvested for FACS analysis. Depletion of DDB2 from fibroblasts was confirmed by western blot analysis. Error bars represent the s.d. ** P < 0.01, n.s., not significant.
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    1) Product Images from "The deacetylase SIRT6 promotes the repair of UV-induced DNA damage by targeting DDB2"

    Article Title: The deacetylase SIRT6 promotes the repair of UV-induced DNA damage by targeting DDB2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa661

    In response to UV irradiation, SIRT6 interacts with DDB2. ( A ) SIRT6 fails to stimulate NER in XPC-depleted HCA2-hTERT cells. A control vector or vector encoding SIRT6 was cotransfected with UVC-treated pmax-GFP and pDsRed2-N1 into control and XPC-depleted HCA2-hTERT cells. At 72 h post transfection, the cells were harvested for FACS analysis. Error bars represent the s.d. *** P < 0.001, n.s., not significant. (B, C) SIRT6 interacts with DDB2 in vivo . HEK293 cells were transfected with Flag-tagged SIRT6. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the Flag tag, followed by western blot analysis with the indicated antibodies ( B ). HEK293 cells were transfected with HA-tagged DDB2. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the HA tag, followed by western blot analysis ( C ). ( D ) SIRT6 interacts with DDB2 in vitro . Five micrograms of recombinant His-DDB2 and GST or GST-SIRT6 together with 30 μl GST-agarose resin were incubated with GST or GST-SIRT6 in IP buffer for 6 h at 4°C. Western blot analysis was performed with the indicated antibodies. ( E ) A schematic representation of the SIRT6 fragments used in this study is shown. ( F ) A control vector or vectors encoding full-length SIRT6-Flag, SIRT6 ΔC-Flag (1-271 aa), SIRT6 ΔN-Flag (49–355 aa) or the deacetylase core fragment SIRT6 ΔNΔC-Flag (49–271 aa) were cotransfected with DDB2-GFP into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with GFP-Trap (Chromotek), followed by western blot analysis. ( G ) A schematic representation of the DDB2 fragments used in this study is shown. ( H ) Vectors encoding full-length DDB2-GFP, DDB2 fragment 1-GFP (1–100 aa), or DDB2 fragment 2-GFP (101–427 aa) were cotransfected with a SIRT6-Flag-expressing vector into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis. ( I ) SIRT6 fails to stimulate NER in DDB2-depleted HCA2-hTERT cells. HCA2-hTERT cells were transfected with control siRNA or DDB2-specific siRNA twice over a 48-h interval. Afterwards, the HCA2-hTERT cells were transfected with a control vector or a vector expressing SIRT6 and UVC-treated pmax-GFP together with pDsRed2-N1. On day 3 post transfection, the cells were harvested for FACS analysis. Depletion of DDB2 from fibroblasts was confirmed by western blot analysis. Error bars represent the s.d. ** P < 0.01, n.s., not significant.
    Figure Legend Snippet: In response to UV irradiation, SIRT6 interacts with DDB2. ( A ) SIRT6 fails to stimulate NER in XPC-depleted HCA2-hTERT cells. A control vector or vector encoding SIRT6 was cotransfected with UVC-treated pmax-GFP and pDsRed2-N1 into control and XPC-depleted HCA2-hTERT cells. At 72 h post transfection, the cells were harvested for FACS analysis. Error bars represent the s.d. *** P < 0.001, n.s., not significant. (B, C) SIRT6 interacts with DDB2 in vivo . HEK293 cells were transfected with Flag-tagged SIRT6. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the Flag tag, followed by western blot analysis with the indicated antibodies ( B ). HEK293 cells were transfected with HA-tagged DDB2. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against the HA tag, followed by western blot analysis ( C ). ( D ) SIRT6 interacts with DDB2 in vitro . Five micrograms of recombinant His-DDB2 and GST or GST-SIRT6 together with 30 μl GST-agarose resin were incubated with GST or GST-SIRT6 in IP buffer for 6 h at 4°C. Western blot analysis was performed with the indicated antibodies. ( E ) A schematic representation of the SIRT6 fragments used in this study is shown. ( F ) A control vector or vectors encoding full-length SIRT6-Flag, SIRT6 ΔC-Flag (1-271 aa), SIRT6 ΔN-Flag (49–355 aa) or the deacetylase core fragment SIRT6 ΔNΔC-Flag (49–271 aa) were cotransfected with DDB2-GFP into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with GFP-Trap (Chromotek), followed by western blot analysis. ( G ) A schematic representation of the DDB2 fragments used in this study is shown. ( H ) Vectors encoding full-length DDB2-GFP, DDB2 fragment 1-GFP (1–100 aa), or DDB2 fragment 2-GFP (101–427 aa) were cotransfected with a SIRT6-Flag-expressing vector into HEK 293 cells. At 24 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis. ( I ) SIRT6 fails to stimulate NER in DDB2-depleted HCA2-hTERT cells. HCA2-hTERT cells were transfected with control siRNA or DDB2-specific siRNA twice over a 48-h interval. Afterwards, the HCA2-hTERT cells were transfected with a control vector or a vector expressing SIRT6 and UVC-treated pmax-GFP together with pDsRed2-N1. On day 3 post transfection, the cells were harvested for FACS analysis. Depletion of DDB2 from fibroblasts was confirmed by western blot analysis. Error bars represent the s.d. ** P < 0.01, n.s., not significant.

    Techniques Used: Irradiation, Plasmid Preparation, Transfection, In Vivo, Immunoprecipitation, FLAG-tag, Western Blot, In Vitro, Recombinant, Incubation, Histone Deacetylase Assay, Expressing

    SIRT6 deacetylates DDB2 in response to UV irradiation. ( A ) The interaction between SIRT6 and DDB2 is enhanced in response to UVC irradiation. HEK293 cells with stable Flag-tagged SIRT6 integration were transfected with DDB2-GFP and treated with or without UVC (20 J/m 2 ). Then, cells were harvested at the indicated time points. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by western blot analysis with the indicated antibodies. IPed, immunoprecipitated. ( B ) DDB2 is a target of the deacetylase SIRT6. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6-Flag. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysines (AcK), followed by western blot analysis with the indicated antibodies. ( C ) DDB2 is deacetylated by SIRT6 in vitro . A recombinant DDB2-GFP protein (5 μg) and a SIRT6 WT or H133Y mutant protein (5 μg) purified from HEK293 cells were incubated to allow the deacetylation reaction to occur in HDAC buffer (50 mM Tris-HCl pH 9.0, 4 mM MgCl 2 , 50 mM NaCl, 0.2 mM DTT and 1 mM NAD+) at 30°C for 2.5 h . Then, the reactions were analyzed by western blot analysis using antibodies against AcK, DDB2 and SIRT6. The recombinant DDB2-GFP, SIRT6 WT or SIRT6 H133Y mutant proteins were also resolved on SDS-PAGE gels, followed by Coomassie Blue staining. The gel was stained with Coomassie reagent (ratio, methanol:acetic acid:Coomassie:H 2 O = 45:10:0.25:45) for 2.5 h, followed by washing with a destaining solution (ratio, methanol:acetic acid:H 2 O = 25:8:67). ( D ) The change in the acetylation level of exogenous DDB2 in response to UV irradiation was evaluated. HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 min post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( E ) The changes in the acetylation levels of exogenous DDB2 in control and SIRT6-depleted cells in response to UV irradiation were evaluated. The control and SIRT6-depleted HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 mins post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( F ) The acetylation levels of DDB2 fragments were assessed. HEK293 cells were transfected with vectors encoding the GFP-tagged DDB2 fragments, DDB2 fragment 1-GFP (1–100 aa) or DDB2 fragment 2-GFP (101–427 aa). At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis. The arrow indicates the acetylated DDB2-F1-GFP. ( G ) The Coomassie blue-stained SDS-PAGE gel image shows that DDB2 immunoprecipitated from HEK293 cells with an anti-GFP antibody. HEK293 cells were transfected with a plasmid encoding DDB2-GFP. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against GFP, followed by SDS-PAGE. ( H ) A schematic representation of lysine residues in DDB2 fragment 1 (1–100 aa) is shown. ( I ) K35 and K77 are the two lysine residues that are acetylated in DDB2. DDB2-GFP WT and the indicated DDB2 mutants were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutants. ( J ) The acetylation levels of DDB2-GFP WT and the 2KR mutant were measured. DDB2-GFP WT and DDB2-GFP 2KR were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutant.
    Figure Legend Snippet: SIRT6 deacetylates DDB2 in response to UV irradiation. ( A ) The interaction between SIRT6 and DDB2 is enhanced in response to UVC irradiation. HEK293 cells with stable Flag-tagged SIRT6 integration were transfected with DDB2-GFP and treated with or without UVC (20 J/m 2 ). Then, cells were harvested at the indicated time points. Cell lysates were immunoprecipitated with an anti-Flag antibody, followed by western blot analysis with the indicated antibodies. IPed, immunoprecipitated. ( B ) DDB2 is a target of the deacetylase SIRT6. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6-Flag. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysines (AcK), followed by western blot analysis with the indicated antibodies. ( C ) DDB2 is deacetylated by SIRT6 in vitro . A recombinant DDB2-GFP protein (5 μg) and a SIRT6 WT or H133Y mutant protein (5 μg) purified from HEK293 cells were incubated to allow the deacetylation reaction to occur in HDAC buffer (50 mM Tris-HCl pH 9.0, 4 mM MgCl 2 , 50 mM NaCl, 0.2 mM DTT and 1 mM NAD+) at 30°C for 2.5 h . Then, the reactions were analyzed by western blot analysis using antibodies against AcK, DDB2 and SIRT6. The recombinant DDB2-GFP, SIRT6 WT or SIRT6 H133Y mutant proteins were also resolved on SDS-PAGE gels, followed by Coomassie Blue staining. The gel was stained with Coomassie reagent (ratio, methanol:acetic acid:Coomassie:H 2 O = 45:10:0.25:45) for 2.5 h, followed by washing with a destaining solution (ratio, methanol:acetic acid:H 2 O = 25:8:67). ( D ) The change in the acetylation level of exogenous DDB2 in response to UV irradiation was evaluated. HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 min post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( E ) The changes in the acetylation levels of exogenous DDB2 in control and SIRT6-depleted cells in response to UV irradiation were evaluated. The control and SIRT6-depleted HCA2-hTERT cells were transfected with a vector encoding DDB2-GFP and irradiated with or without UVC (20 J/m 2 ). Then, the cells were harvested at 5 mins post UV irradiation. Cell lysates were immunoprecipitated with GFP-Trap, followed by western blot analysis with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP. ( F ) The acetylation levels of DDB2 fragments were assessed. HEK293 cells were transfected with vectors encoding the GFP-tagged DDB2 fragments, DDB2 fragment 1-GFP (1–100 aa) or DDB2 fragment 2-GFP (101–427 aa). At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis. The arrow indicates the acetylated DDB2-F1-GFP. ( G ) The Coomassie blue-stained SDS-PAGE gel image shows that DDB2 immunoprecipitated from HEK293 cells with an anti-GFP antibody. HEK293 cells were transfected with a plasmid encoding DDB2-GFP. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against GFP, followed by SDS-PAGE. ( H ) A schematic representation of lysine residues in DDB2 fragment 1 (1–100 aa) is shown. ( I ) K35 and K77 are the two lysine residues that are acetylated in DDB2. DDB2-GFP WT and the indicated DDB2 mutants were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutants. ( J ) The acetylation levels of DDB2-GFP WT and the 2KR mutant were measured. DDB2-GFP WT and DDB2-GFP 2KR were transfected into HEK293 cells. GFP-tagged DDB2 was pulled down from cell lysates using an anti-acetylated lysine antibody and immunoblotted with the indicated antibodies. The arrow indicates the acetylated DDB2-GFP WT or mutant.

    Techniques Used: Irradiation, Transfection, Immunoprecipitation, Western Blot, Histone Deacetylase Assay, In Vitro, Recombinant, Mutagenesis, Purification, Incubation, SDS Page, Staining, Plasmid Preparation

    Loss of SIRT6 leads to the retention of DDB2 on chromatin in response to UV irradiation. ( A ) Changes in DDB2 bound to chromatin in the absence of SIRT6 in response to UV irradiation were evaluated. Control and SIRT6-depleted HCA2-hTERT cells were irradiated with UVC (20 J/m 2 ), harvested at the indicated time points and subjected to cellular protein fractionation, followed by western blot analysis of DDB2 in the chromatin fractions. ( B ) Depleting SIRT6 abolished the increased ubiquitination of DDB2 upon UV irradiation. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag and treated with MG132 at 10 μM. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( C ) The ubiquitination level of the DDB2 2KR mutant was lower than that of DDB2 WT. HEK293 cells were transfected with a DDB2-Flag or DDB2-2KR-Flag plasmid. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( D ) DDB2 2KR partially abolished the interaction between DDB2 and p97. HEK293 cells were transfected with a pControl, DDB2-GFP or DDB2-2KR-GFP plasmid. After 16 h, the cells were irradiated with UVC or left untreated, and at 5 min post UV irradiation, the cells were harvested for immunoprecipitation with an antibody against GFP and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( E ) SIRT6 greatly diminished the interaction between DDB2 and p97. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( F ) Immunoblot analysis of changes in XPC hyperubiquitination in the absence or presence of SIRT6 was performed. Control and SIRT6-depleted HCA2-hTERT cells were UVC irradiated (20 J/m 2 ) and harvested at the indicated time points, followed by western blot analysis.
    Figure Legend Snippet: Loss of SIRT6 leads to the retention of DDB2 on chromatin in response to UV irradiation. ( A ) Changes in DDB2 bound to chromatin in the absence of SIRT6 in response to UV irradiation were evaluated. Control and SIRT6-depleted HCA2-hTERT cells were irradiated with UVC (20 J/m 2 ), harvested at the indicated time points and subjected to cellular protein fractionation, followed by western blot analysis of DDB2 in the chromatin fractions. ( B ) Depleting SIRT6 abolished the increased ubiquitination of DDB2 upon UV irradiation. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag and treated with MG132 at 10 μM. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( C ) The ubiquitination level of the DDB2 2KR mutant was lower than that of DDB2 WT. HEK293 cells were transfected with a DDB2-Flag or DDB2-2KR-Flag plasmid. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag, followed by western blot analysis with an antibody recognizing ubiquitin. ( D ) DDB2 2KR partially abolished the interaction between DDB2 and p97. HEK293 cells were transfected with a pControl, DDB2-GFP or DDB2-2KR-GFP plasmid. After 16 h, the cells were irradiated with UVC or left untreated, and at 5 min post UV irradiation, the cells were harvested for immunoprecipitation with an antibody against GFP and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( E ) SIRT6 greatly diminished the interaction between DDB2 and p97. Control and SIRT6-depleted HEK293 cells were transfected with a plasmid encoding DDB2-Flag. After 16 h, the cells were harvested for immunoprecipitation with an antibody against Flag and immunoblotted with the indicated antibodies. IPed, immunoprecipitated. ( F ) Immunoblot analysis of changes in XPC hyperubiquitination in the absence or presence of SIRT6 was performed. Control and SIRT6-depleted HCA2-hTERT cells were UVC irradiated (20 J/m 2 ) and harvested at the indicated time points, followed by western blot analysis.

    Techniques Used: Irradiation, Fractionation, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis

    Several mutations in SIRT6 in melanoma impair the repair of UV-induced DNA damage and result in a high incidence of mutation rates across the genome. ( A ) Lolliplot of the SIRT6 protein with the alterations present in melanoma samples indicated. ( B ) Locations of the alterations mapped to the SIRT6 crystal structure (PDB: 3ZG6). ( C ) Number of nonsynonymous mutations in melanoma samples with SIRT6 mutations. ( D ) Several SIRT6 mutants lost the ability to enhance NER efficiency. Error bars represent the s.d. *** P < 0.001, ** P < 0.01, n.s., not significant. ( E ) The SIRT6 P27S and H50Y mutants partially lost their deacetylase activity. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6 WT or mutants. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis with the indicated antibodies. ( F ) SIRT6 G134W has a high turnover rate. HCA2-hTERT cells were harvested for protein extraction at 3 and 24 h post transfection with a control vector, SIRT6 WT or G134W mutant, followed by western blot analysis of SIRT6 expression.
    Figure Legend Snippet: Several mutations in SIRT6 in melanoma impair the repair of UV-induced DNA damage and result in a high incidence of mutation rates across the genome. ( A ) Lolliplot of the SIRT6 protein with the alterations present in melanoma samples indicated. ( B ) Locations of the alterations mapped to the SIRT6 crystal structure (PDB: 3ZG6). ( C ) Number of nonsynonymous mutations in melanoma samples with SIRT6 mutations. ( D ) Several SIRT6 mutants lost the ability to enhance NER efficiency. Error bars represent the s.d. *** P < 0.001, ** P < 0.01, n.s., not significant. ( E ) The SIRT6 P27S and H50Y mutants partially lost their deacetylase activity. HEK293 cells were cotransfected with plasmids encoding DDB2-GFP and SIRT6 WT or mutants. At 16 h post transfection, the cells were harvested for immunoprecipitation with an antibody against acetylated lysine, followed by western blot analysis with the indicated antibodies. ( F ) SIRT6 G134W has a high turnover rate. HCA2-hTERT cells were harvested for protein extraction at 3 and 24 h post transfection with a control vector, SIRT6 WT or G134W mutant, followed by western blot analysis of SIRT6 expression.

    Techniques Used: Mutagenesis, Histone Deacetylase Assay, Activity Assay, Transfection, Immunoprecipitation, Western Blot, Protein Extraction, Plasmid Preparation, Expressing

    rabbit anti dna binding protein 2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti dna binding protein 2
    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; <t>DDB2,</t> DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.
    Rabbit Anti Dna Binding Protein 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells"

    Article Title: Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00160.2018

    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.
    Figure Legend Snippet: p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.

    Techniques Used: Immunolabeling, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.
    Figure Legend Snippet: p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.

    Techniques Used: Western Blot, Binding Assay, Knock-Out, Immunolabeling, Staining

    rabbit anti dna binding protein 2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti dna binding protein 2
    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; <t>DDB2,</t> DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.
    Rabbit Anti Dna Binding Protein 2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells"

    Article Title: Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00160.2018

    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.
    Figure Legend Snippet: p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.

    Techniques Used: Immunolabeling, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.
    Figure Legend Snippet: p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.

    Techniques Used: Western Blot, Binding Assay, Knock-Out, Immunolabeling, Staining

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    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; <t>DDB2,</t> DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.
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    USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

    doi: 10.3389/fcell.2021.663411

    Figure Lengend Snippet: USP44 deubiquitinates DDB2 following UVC exposure. (A,B) MEFs of the indicated genotype were exposed to 30 J/m 2 UVC and samples were collected for immunoblot at the indicated times. The doublet bands were consistently observed in these mouse cells, and were both quantitated to represent DDB2 using imageJ. The graph represents the mean ± SEM for three independent MEF lines. “*” denotes p < 0.05. (C) MEFs were transduced with the indicated constructs and subjected to immunoprecipitation for the FLAG epitope and probed as indicated. (D) Purified CRL-DDB2 was auto-ubiquitinated by the addition of UbE1, UBCH5a, and ubiquitin, and subsequently incubated with either wild type or catalytic mutant (C281A) immunopurified USP44 as indicated. Rxn = reaction.

    Article Snippet: Antibodies used in this study include anti-DDB2 (#5416), FLAG (DYKDDDDK, #2368), histone H2B (#5546), α-actin (#4970) (Cell Signaling Inc. Danvers, MA, United States), HA (3F10, Millipore-Sigma, St. Louis, MO, United States), V5 (Bethyl Laboratories, Montgomery, TX, United States), CPD (clone TDM-2; CosmoBio) XPC (graciously supplied by Jeff Salisbury) ( ).

    Techniques: Western Blot, Transduction, Construct, Immunoprecipitation, FLAG-tag, Purification, Incubation, Mutagenesis

    Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: USP44 Stabilizes DDB2 to Facilitate Nucleotide Excision Repair and Prevent Tumors

    doi: 10.3389/fcell.2021.663411

    Figure Lengend Snippet: Incomplete CPD repair in Usp44 null cells associated with inadequate DDB2 recruitment to sites of damage. (A,B) VH10 cells expressing DDB2-GFP were transfected with control or USP44 targeting siRNA and then locally irradiated. The DDB-GFP fluorescence was monitored over time using live-cell confocal imaging and quantified to pre-damage intensity set at 100. The graph represents the mean ± SEM for 31–36 cells per condition. (C) MEFs of the indicated genotypes were transduced with wild-type, catalytic mutant (USP44 C I ; C281A), centrin-binding deficient (USP44 C BM ; W162A) USP44, or DDB2 as indicated. The cells were exposed to UVC (10 J/m 2 ) and CPD levels were monitored at the indicated times. The graph represents the means of three independent experiments for each condition.

    Article Snippet: Antibodies used in this study include anti-DDB2 (#5416), FLAG (DYKDDDDK, #2368), histone H2B (#5546), α-actin (#4970) (Cell Signaling Inc. Danvers, MA, United States), HA (3F10, Millipore-Sigma, St. Louis, MO, United States), V5 (Bethyl Laboratories, Montgomery, TX, United States), CPD (clone TDM-2; CosmoBio) XPC (graciously supplied by Jeff Salisbury) ( ).

    Techniques: Expressing, Transfection, Irradiation, Fluorescence, Imaging, Transduction, Mutagenesis, Binding Assay

    p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

    doi: 10.1152/ajpheart.00160.2018

    Figure Lengend Snippet: p53 transactivation domain (TAD) deletion does not affect pluripotent markers in human induced pluripotent stem cells (hiPSCs). A: representative images of Oct4-, Sox2-, and Nanog-immunolabeled (red) p53-TAD knockout (KO) hiPSCs. Phalloidin is shown in green; the nucleus is shown in blue. B−D: mRNA transcript levels of pluripotent markers (B), p53-regulated genes (C), and p53-regulated DNA repair genes (D) in wild-type (WT) and p53-TAD KO hiPSCs as measured by RT-PCR. Symbols denote plotted values. Data are means ± SD; n = 3. *P < 0.05 vs. WT. RQ, relative quantity; a.u., arbitrary units; CDKN1A, cyclin-dependent kinase inhibitor 1A; PIDD, p53-induced death domain; XPA, xeroderma pigmentosum group A; DDB2, DNA-binding protein 2; IGF1R, insulin-like growth factor 1 receptor; DNA Pol η, DNA polymerase H; XPD, xeroderma pigmentosum group D; BBC3, Bcl-2-binding component 3.

    Article Snippet: The following antibodies were used: NH 2 -terminal-specific rabbit monoclonal anti-p53 (Cell Signaling Technology, Danvers, MA), rabbit COOH-terminal anti-p53 (Sigma-Aldrich), rabbit monoclonal anti-checkpoint kinase 2 (Chk2; Thr 68 , Cell Signaling Technology), rabbit monoclonal anti-Chk2 (Cell Signaling Technology), rabbit monoclonal anti-Mre11 (Cell Signaling Technology), rabbit anti-Rad50 (Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group D (XPD; D3Z6I, Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group A (XPA; D9U5U, Cell Signaling Technology), rabbit anti-DNA polymerase H (DNA Pol η; E1I7T, Cell Signaling Technology), and rabbit anti-DNA-binding protein 2 (DDB2; D4C4, Cell Signaling Technology).

    Techniques: Immunolabeling, Knock-Out, Reverse Transcription Polymerase Chain Reaction, Binding Assay

    p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    Article Title: Transactivation domain of p53 regulates DNA repair and integrity in human iPS cells

    doi: 10.1152/ajpheart.00160.2018

    Figure Lengend Snippet: p53 transactivation domain (TAD) deletion impairs DNA damage repair in human induced pluripotent stem cells (hiPSCs). A and B, left: Western blots of Rad50, Mre11, phosphorylated checkpoint kinase 2 (p-Chk2) at Thr68, and total Chk2 (A) as well as xeroderma pigmentosum group D (XPD), xeroderma pigmentosum group A (XPA), DNA-binding protein 2 (DDB2), and DNA polymerase H (DNA Pol η) (B) in wild-type (WT) and p53-TAD knockout (KO) hiPSCs with and without doxorubicin (Doxo) treatment. Western blot quantifications are shown on the right. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. the respective nontreated cells; †P < 0.05 vs. WT; ‡P < 0.05 vs. WT + Doxo. C: p53-TAD hiPSCs were immunolabeled for γH2A.X (green; left) and counterstained with DAPI (blue; right). D: schematic of DNA damage repair methodology. E: nucleoids in WT hiPSCs and p53-TAD KO hiPSCs treated with Doxo (top) and after recovery (bottom) were stained with Vista green dye (green). Comets were apparent with Doxo and after recovery of p53-TAD KO hiPSCs, whereas intact DNA was noted in WT hiPSCs after recovery. F: tail moment of WT hiPSCs and p53-TAD KO hiPSCs nuclei at baseline (control), after Doxo, and after recovery. a.u., arbitrary units. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. control; **P < 0.05 vs. Doxo. G: representative micrograph of hiPSCs immunolabeled for γH2A.X (green; left) and nuclei stained with DAPI (blue; right). H: fraction of WT p53 and p53-TAD KO hiPSCs positive for γH2A.X. Symbols denote plotted values. Data are means ± SD; n = 3 in all cases. *P < 0.05 vs. WT.

    Article Snippet: The following antibodies were used: NH 2 -terminal-specific rabbit monoclonal anti-p53 (Cell Signaling Technology, Danvers, MA), rabbit COOH-terminal anti-p53 (Sigma-Aldrich), rabbit monoclonal anti-checkpoint kinase 2 (Chk2; Thr 68 , Cell Signaling Technology), rabbit monoclonal anti-Chk2 (Cell Signaling Technology), rabbit monoclonal anti-Mre11 (Cell Signaling Technology), rabbit anti-Rad50 (Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group D (XPD; D3Z6I, Cell Signaling Technology), rabbit anti-xeroderma pigmentosum group A (XPA; D9U5U, Cell Signaling Technology), rabbit anti-DNA polymerase H (DNA Pol η; E1I7T, Cell Signaling Technology), and rabbit anti-DNA-binding protein 2 (DDB2; D4C4, Cell Signaling Technology).

    Techniques: Western Blot, Binding Assay, Knock-Out, Immunolabeling, Staining