Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Antibodies against tubulin (TU-02; sc-8035), c-myc (9E10; sc-40), and p53 (DO-1; sc-126) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing FLAG epitope (#14793), BCAT1 (#88785), KU-80 (#2180), KU-70 (#4588), phosphorylated H2AX (pS139; #9718), phosphorylated DNA-PKcs (pS2056; #68716), total DNA-PKcs (#12311), phosphorylated ATM (pS1981; #5883), total ATM (#2873), phosphorylated CHK1 (pS345; #2348), phosphorylated CHK2 (pT68; #2197), total CHK2 (#6334), phosphorylated TP53 (pS15; #12571), and GADPH (#5174) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CRL4 is a major regulator of CHK1 stability. (A and B) Contributions made by CRL1 and CRL4 to CHK1 stability. HeLa cells transfected with control siRNA (GL3) or siRNAs specific for either CUL1 or CUL4 were cultured for 48 h and then incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 3 independent experiments is shown in panel B. (C) Effects of UV on CHK1 stability. HeLa cells transfected with control siRNA or siRNAs specific for CUL1 or CUL4 were mock irradiated or exposed to 50 J/m2 UV at 48 h posttransfection. Cells were harvested 6 h later and analyzed by Western blotting. Levels of CHK1 in knockdown cells are shown relative to levels in control cells (lane 1). (D) Interactions between CUL4A and CHK1. The Flag-tagged CHK1 WT or the CHK1 3RE or CHK2 SA mutant was coproduced with V5-tagged CUL1 or CUL4A in HeLa cells. Cell lysates were resolved directly by SDS-PAGE or were immunoprecipitated with anti-Flag M2 affinity gel prior to SDS-PAGE. Whole-cell lysates (WCL) and immunoprecipitates (IP: Flag) were then analyzed by Western blotting. CUL1 and CUL4A were detected by using an anti-V5 antibody.

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Transfection, Control, Cell Culture, Incubation, Western Blot, Quantitation Assay, Irradiation, Knockdown, Mutagenesis, SDS Page, Immunoprecipitation

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CDT2 is a novel CHK1 regulator. (A and B) CDT2 is required for CHK1 turnover. HeLa cells transfected with control siRNA (GL3) or pooled siRNAs specific for CDT2 were cultured for 48 h and then incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 3 independent experiments is shown in panel B (N.S., not statistically significant by Student's t test). (C) Regulation of CHK1 by CDT2. HeLa cells transfected with control siRNA or individual siRNAs specific for CDT2 were cultured for 48 h and then incubated in the presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. (D) Effects of HU on CHK1 turnover. HeLa cells transfected with control siRNA or siRNA specific for CDT2 were cultured for 48 h and then incubated in the presence of the indicated doses of HU for 6 h. Cell lysates were analyzed by Western blotting. (E) CHK1 levels after dephosphorylation in vitro. HeLa cells transfected with control siRNA or siRNAs specific for CDT2 were cultured for 48 h and then incubated in medium containing 5 mM HU for 6 h. Cell lysates were incubated in the absence or presence of Lambda protein phosphatase (PPase), followed by Western blotting. Levels of CHK1 in cells knocked down for CDT2 are shown relative to levels in control cells (lanes 1 and 3). (F) Effects of UV on CHK1 turnover. HeLa cells transfected with control siRNA or siRNA specific for CDT2 were mock irradiated or exposed to 50 J/m2 UV at 48 h posttransfection. Cells were harvested 6 h later and analyzed by Western blotting. Levels of CHK1 in knockdown cells are shown relative to levels in control cells (lane 1). (G) Regulation of CHK1 stability by CRL1, CRL4, and CDT2. HeLa cells transfected with control siRNA or siRNAs specific for CUL1, CUL4, or CDT2 were cultured for 48 h and then incubated in the presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. Levels of CHK1 in knockdown cells are shown relative to levels in control cells (lane 1).

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Transfection, Control, Cell Culture, Incubation, Western Blot, Quantitation Assay, De-Phosphorylation Assay, In Vitro, Irradiation, Knockdown

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CDT2 regulates CHK1 turnover in response to replication stress. (A and B) HU-induced CHK1 turnover in CDT2-depleted cells. HeLa cells transfected with control siRNA (GL3) or siRNA specific for CDT2 were cultured for 48 h and then incubated in medium containing 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 5 independent experiments is shown in panel B. (C and D) UV-induced CHK1 turnover in CDT2-depleted cells. HeLa cells transfected with control siRNA or siRNA specific for CDT2 were exposed to 50 J/m2 UV at 48 h posttransfection. Cells were then cultured in the absence or presence of 10 μM MG132. At the indicated times post-UV exposure, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel C, and the quantitation of data from 4 independent experiments is shown in panel D. (E) Prolonged half-life of CHK1 in CDT2-depleted cells. HeLa Tet-on cells were transfected with a plasmid encoding Tet-inducible Flag-CHK1 followed by either control siRNA or siRNA specific for CDT2. Cells were cultured in the presence of 0.2 μg/ml doxycycline for 14 h to induce Flag-CHK1 expression. Cells were washed, cultured for 4 h in fresh medium, and then cultured in medium containing 5 mM HU. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. Levels of Flag-CHK1 are shown relative to levels at the 0-h time points (lanes 1 and 4). (F) Effects of CDT1 overexpression on CHK1 stability. HeLa cells expressing Flag-tagged CDT1 for 24 h were incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. The arrowhead indicates endogenous CDT1.

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Transfection, Control, Cell Culture, Incubation, Western Blot, Quantitation Assay, Plasmid Preparation, Expressing, Over Expression

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CHK1 maintains G2 arrest in CDT2-depleted cells. (A and B) Stabilization of the active form of CHK1 in CDT2-depleted cells. HeLa cells transfected with control siRNA (GL3) or siRNA specific for CDT2 were cultured for 48 h and then incubated in medium containing dimethyl sulfoxide (DMSO) or 30 nM AZD7762 (AZD) for 2 h. Cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 3 independent experiments is shown in panel B. (C and D) G2 checkpoint regulation by CHK1 and CDT2. HeLa cells transfected with control siRNA or siRNA specific for CDT2 were cultured for 48 h and then incubated in medium containing dimethyl sulfoxide or 30 nM AZD7762 for 4 h. Cells were analyzed by flow cytometry. A representative experiment is shown in panel C, and the quantitation of data from 3 independent experiments is shown in panel D.

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Transfection, Control, Cell Culture, Incubation, Western Blot, Quantitation Assay, Flow Cytometry

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CDT2 serves as the substrate receptor that targets CHK1 to CRL4CDT2. (A) Interactions between CHK1 and CDT2. Flag-tagged WT or a mutant form (3RE) of CHK1 was coproduced with Myc-tagged CDT2 in HeLa cells for 24 h. Cells were then cultured in medium containing 5 mM HU and 10 μM MG132 for 4 h. Cell lysates were resolved directly by SDS-PAGE or were immunoprecipitated with anti-Flag M2 affinity gel prior to SDS-PAGE. Whole-cell lysates (WCL) and immunoprecipitates (IP: Flag) were then analyzed by Western blotting. (B) Subcellular localization of CHK1 and CDT2. Flag-tagged CHK1 was produced in HeLa cells for 48 h. Cells were then cultured in the absence or presence of 5 mM HU or 10 μM MG132 for 4 h. The localization of endogenous CDT2 and ectopic CHK1 was determined by immunofluorescence staining. (C) Interactions between CHK1 and CRL4CDT2. Flag-tagged CHK1 was coproduced with the Myc-tagged CDT2 WT or CDT2 RA mutant in HeLa cells for 24 h. Cells were exposed to 50 J/m2 UV and cultured in the presence of 10 μM MG132 for 2 h. Cell lysates were resolved directly by SDS-PAGE or were immunoprecipitated with anti-Flag M2 affinity gel prior to SDS-PAGE. Whole-cell lysates and immunoprecipitates were then analyzed by Western blotting. (D) Regulation of CDT2 levels in response to replication stress. HeLa cells were cultured in medium containing 50 μg/ml cycloheximide (CHX), 5 mM HU, or both. At the indicated times post-HU/CHX treatment, cells were harvested and analyzed by Western blotting. (E and F) CHK1 ubiquitination in vitro. HeLa cells transfected with control siRNA (GL3) or siRNAs specific for either CDT2 or DDB1 were cultured for 48 h and then incubated in the presence of 10 mM HU for 1 h. Cell lysates were prepared, and a portion was resolved directly by SDS-PAGE followed by Western blotting (E). The remaining lysates were immunoprecipitated with anti-CUL4A antibody. Immunoprecipitated CRL4 complexes were then incubated with purified E1, E2, ubiquitin, and Flag-CHK1 that had been purified from HU-treated cells (F). Long (L) and short (S) exposures are indicated.

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Mutagenesis, Cell Culture, SDS Page, Immunoprecipitation, Western Blot, Produced, Immunofluorescence, Staining, Ubiquitin Proteomics, In Vitro, Transfection, Control, Incubation, Purification

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: Claspin binding is important for CHK1 regulation by CRL4CDT2. (A) Schematic representation of CHK1 indicating the domain structure as well as kinase-inactive (A36F), claspin binding (K54A, R129A, and T153A), and SQ/TQ phosphorylation site (3SA) mutants. (B) Role of kinase activity and claspin binding in mediating interactions between CHK1 and CDT2. Flag-tagged WT or mutant forms of CHK1 were coproduced with Myc-tagged CDT2 for 24 h and then cultured in medium containing 5 mM HU and 10 μM MG132 for 4 h. Cell lysates were resolved directly by SDS-PAGE or were immunoprecipitated with anti-Flag M2 affinity gel prior to SDS-PAGE. Whole-cell lysates (WCL) and immunoprecipitates (IP: Flag) were analyzed by Western blotting. (C and D) Contributions made by kinase activity and claspin binding to CHK1 turnover. HeLa cells expressing the CHK1 WT or the indicated CHK1 mutants were cultured in medium containing 10 μg/ml CHX and 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU/CHX treatment, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel C, and the quantitation of data from 4 independent experiments is shown in panel D. (E) Role of ATR-mediated phosphorylation in CHK1 turnover. HeLa cells expressing the CHK1 WT or the CHK1 3SA mutant were cultured in medium containing 10 μg/ml CHX and 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU/CHX treatment, cells were harvested and analyzed by Western blotting. Levels of Flag-CHK1 are shown relative to levels at the 0-h time points.

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Binding Assay, Phospho-proteomics, Activity Assay, Mutagenesis, Cell Culture, SDS Page, Immunoprecipitation, Western Blot, Expressing, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CRL4CDT2 targets CHK1 for PCNA-independent degradation. (A and B) Role of PCNA binding in regulating CHK1 stability. HeLa cells transfected with control siRNA (GL3) or siRNA specific for PCNA were cultured for 48 h and then incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. A representative experiment is shown in panel A, and the quantitation of data from 3 independent experiments is shown in panel B. (C) PCNA binding is not required for CHK1 turnover in UV-treated cells. HeLa cells transfected with control siRNA or siRNA specific for PCNA were mock irradiated or exposed to 50 J/m2 UV at 48 h posttransfection. Cells were harvested 6 h later and analyzed by Western blotting. Levels of CHK1 in knockdown cells are shown relative to levels in control cells (lane 1). (D and E) HU-induced CHK1 turnover in PCNA-depleted cells. HeLa cells transfected with control siRNA or siRNA specific for PCNA were cultured for 48 h and then incubated in medium containing 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU treatment, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel D, and the quantitation of data from 4 independent experiments is shown in panel E. (F and G) PCNA is required for CDT1 and SET8 but not CHK1 turnover. HeLa cells transfected with control siRNA, siRNA specific for CUL1, or siRNAs specific for CUL1 and PCNA were cultured for 48 h and then incubated in the absence or presence of 5 mM HU for 6 h. Cell lysates were analyzed by Western blotting. A representative experiment is shown in panel F, and the quantitation of data from 3 independent experiments is shown in panel G. In panels B, E, and G, differences were not significant between control cells and PCNA, CUL1, or CUL1/PCNA knockdown cells within either treatment condition (Student's t test).

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Binding Assay, Transfection, Control, Cell Culture, Incubation, Western Blot, Quantitation Assay, Irradiation, Knockdown

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CHK1 PIP boxes are dispensable for CRL4CDT2-mediated destruction. (A) Schematic representation of CHK1 indicating the domain organization. The putative N-terminal PIP degron (NPIP box), C-terminal PIP box (CPIP box), and PIP box mutants (ΔNPIP1, ΔNPIP2, and 2FA) are shown. Conserved residues in the canonical PIP box and PIP degron are shown in black and dark gray, respectively. Additional residues that are highly conserved in CHK1 PIP boxes are shown in dark gray. (B) Role of the CPIP box in mediating CDT2 binding. The indicated Flag-tagged proteins were coproduced with Myc-tagged CDT2 in HeLa cells for 24 h and then cultured in medium containing 5 mM HU and 10 μM MG132 for 4 h. Cell lysates were resolved directly by SDS-PAGE or were incubated with anti-Flag M2 affinity gel prior to SDS-PAGE. Whole-cell lysates (WCL) and immunoprecipitates (IP: Flag) were analyzed by Western blotting. Levels of pS296 were normalized to levels of precipitated Flag-CHK1 and are shown relative to those of the CHK1 WT. (C and D) Role of the CPIP box in regulating CHK1 stability. HeLa cells expressing the CHK1 WT or the CHK1 2FA mutant were cultured in medium containing 10 μg/ml CHX and 5 mM HU in the absence or presence of 10 μM MG132. At the indicated times post-HU/CHX treatment, cells were harvested and analyzed by Western blotting. A representative experiment is shown in panel C, and the quantitation of data from 3 independent experiments is shown in panel D. Long (L) and short (S) exposures are indicated. (E) Role of the CHK1 NPIP box in mediating CDT2 binding. The indicated Flag-tagged proteins were coproduced with Myc-tagged CDT2 in HeLa cells for 24 h and then cultured in medium containing 5 mM HU and 10 μM MG132 for 4 h. Cell lysates were resolved directly by SDS-PAGE or were incubated with anti-Flag M2 affinity gel prior to SDS-PAGE. Whole-cell lysates and immunoprecipitates were analyzed by Western blotting. Levels of pS296 were normalized to levels of precipitated Flag-CHK1 and are shown relative to those of the CHK1 WT. (F and G) Role of the NPIP box in regulating CHK1 stability. HeLa cells expressing the CHK1 3RE or 3RE ΔNPIP2 mutant were cultured in medium containing 10 μg/ml CHX and 5 mM HU. Cells were harvested and analyzed by Western blotting at the indicated times posttreatment. A representative experiment is shown in panel F, and the quantitation of data from 3 independent experiments is shown in panel G.

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Binding Assay, Cell Culture, SDS Page, Incubation, Western Blot, Expressing, Mutagenesis, Quantitation Assay

Journal: Molecular and Cellular Biology

Article Title: CRL4 CDT2 Targets CHK1 for PCNA-Independent Destruction

doi: 10.1128/MCB.00847-12

Figure Lengend Snippet: CRL4CDT2 interacts with CHK1 in the nucleoplasm. (A) Spatial regulation of CHK1 ubiquitination by CRL1 and CRL4. HeLa cells were exposed to 20 J/m2 UV and then harvested at the indicated time points post-UV exposure. Subcellular fractionation was performed, and individual fractions were analyzed by Western blotting. The arrowheads indicate the neddylated form of cullins. (B) Subcellular location of CHK1 interactions with CDT2. HeLa cells were exposed to 50 J/m2 UV. Cytoplasmic (C), soluble nuclear (N), and chromatin (CH) fractions were isolated 2 h later and either resolved directly by SDS-PAGE (input) or incubated with protein A-agarose either alone (IP: Control) or with affinity-purified anti-CHK1 antibody (IP: CHK1) prior to SDS-PAGE. (C) The checkpoint activation/termination cycle. In response to replication stress, CHK1 is recruited to sites of DNA damage in a claspin-dependent manner, where it is phosphorylated by ATR. Phosphorylated CHK1 is then released from the chromatin to phosphorylate downstream effectors. Two E3 ubiquitin ligases target CHK1 for destruction. CRL4CDT2 ubiquitinates CHK1 in the nucleoplasm, whereas CRL1FBX6 ubiquitinates CHK1 in the cytoplasm. This terminates checkpoint signaling through the ATR/CHK1 pathway.

Article Snippet: The following antibodies were purchased: CHK1 (G-4), CUL1 (H-213), CDT2 (B-8), Myc (9E10), PCNA (PC10), SKP1, and FBX6 (7B11) antibodies were purchased from Santa Cruz Biotechnology; actin, Flag (M2), and claspin antibodies were purchased from Sigma; CUL4 and CDT1 antibodies were purchased from Bethyl Laboratories; CUL4A antibody was purchased from Rockland Immunochemicals; V5, CDT2, DDB1, and tubulin antibodies were purchased from Abcam; CHK1 phospho-S296 (pS296) antibody was purchased from Epitomics; CHK1 phospho-S345 (pS345) (133D3) antibody was purchased from Cell Signaling; β-catenin and ORC2 antibodies were purchased from BD Biosciences; and SET8 antibody was purchased from Millipore.

Techniques: Ubiquitin Proteomics, Fractionation, Western Blot, Isolation, SDS Page, Incubation, Control, Affinity Purification, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

doi: 10.1083/jcb.201003004

Figure Lengend Snippet: DNA damage fails to activate Chk1 or mitotic delay in late G2–arrested Cdk1AS or HCT116 cells. (A) Mitotic entry in Cdk1AS cells released from 1NM-PP1 arrest with or without irradiation. (B) γ-H2AX staining of 1NM-PP1–arrested Cdk1AS cells before and after release with or without irradiation. (C) Western blot of total and pS345 Chk1 in asynchronous (Asy) and 1NM-PP1–arrested Cdk1AS cells 30 min after exposure to 10 Gy ionizing radiation (IR) and after release. (D–F) As for A–C using RO-3306–arrested HCT116 colon carcinoma cells. (A and D) Values represent the mean and standard deviation of three independent experiments. Ctrl, control. Bars, 10 µm.

Article Snippet: Monoclonal antibodies against Chk1 (G-4) and actin (C-2) and polyclonal antibodies against Cdc2 phosphorylated at Y15 were purchased from Santa Cruz Biotechnology, Inc. Polyclonal antibodies against pS345 Chk1 and Mre11 were obtained from Cell Signaling Technology, whereas monoclonal antibody against α-tubulin (DM1A) was obtained from Sigma-Aldrich.

Techniques: Irradiation, Staining, Western Blot, Standard Deviation, Control

Journal: The Journal of Cell Biology

Article Title: Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

doi: 10.1083/jcb.201003004

Figure Lengend Snippet: DNA damage–induced Chk1 S345 phosphorylation can be restored in late G2–arrested cells by Akt inhibition. (A) Comparison of TopBP1-mediated activation of ATR and Chk1 pS345 phosphorylation in asynchronous (Asy) and 1NM-PP1–arrested Cdk1AS cells exposed to tamoxifen or irradiation. (B) Western blot of asynchronous or G2-arrested Cdk1AS cells pretreated with the indicated kinase inhibitors for 1.5 h plus or minus irradiation. (C) Western blot of G2-arrested HCT116 cells pretreated with Akt inhibitor for 1.5 h plus or minus irradiation. IR, ionizing radiation.

Article Snippet: Monoclonal antibodies against Chk1 (G-4) and actin (C-2) and polyclonal antibodies against Cdc2 phosphorylated at Y15 were purchased from Santa Cruz Biotechnology, Inc. Polyclonal antibodies against pS345 Chk1 and Mre11 were obtained from Cell Signaling Technology, whereas monoclonal antibody against α-tubulin (DM1A) was obtained from Sigma-Aldrich.

Techniques: Phospho-proteomics, Inhibition, Comparison, Activation Assay, Irradiation, Western Blot

Journal: The Journal of Cell Biology

Article Title: Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

doi: 10.1083/jcb.201003004

Figure Lengend Snippet: PTEN inactivation suppresses DNA damage signaling. (A) Western blot analysis of total and pS345 Chk1, pS473 Akt, PTEN, Mre11, CtIP, and tubulin expression in parental HCT116 cells and neomycin-resistant and PTEN-deficient derivatives. (B) Subcellular localization of RPA, CtIP, and γ-H2AX in parental or PTEN-deficient HCT116 cells plus or minus irradiation (10 Gy). DAPI staining is included in the merged images to visualize nuclei. IR, ionizing radiation. Bars, 10 µm.

Article Snippet: Monoclonal antibodies against Chk1 (G-4) and actin (C-2) and polyclonal antibodies against Cdc2 phosphorylated at Y15 were purchased from Santa Cruz Biotechnology, Inc. Polyclonal antibodies against pS345 Chk1 and Mre11 were obtained from Cell Signaling Technology, whereas monoclonal antibody against α-tubulin (DM1A) was obtained from Sigma-Aldrich.

Techniques: Western Blot, Expressing, Irradiation, Staining

Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Antibodies against tubulin (TU-02; sc-8035), c-myc (9E10; sc-40), and p53 (DO-1; sc-126) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing FLAG epitope (#14793), BCAT1 (#88785), KU-80 (#2180), KU-70 (#4588), phosphorylated H2AX (pS139; #9718), phosphorylated DNA-PKcs (pS2056; #68716), total DNA-PKcs (#12311), phosphorylated ATM (pS1981; #5883), total ATM (#2873), phosphorylated CHK1 (pS345; #2348), phosphorylated CHK2 (pT68; #2197), total CHK2 (#6334), phosphorylated TP53 (pS15; #12571), and GADPH (#5174) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay

Journal: The Journal of Cell Biology

Article Title: Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2

doi: 10.1083/jcb.201003004

Figure Lengend Snippet: PTEN inactivation suppresses DNA damage signaling. (A) Western blot analysis of total and pS345 Chk1, pS473 Akt, PTEN, Mre11, CtIP, and tubulin expression in parental HCT116 cells and neomycin-resistant and PTEN-deficient derivatives. (B) Subcellular localization of RPA, CtIP, and γ-H2AX in parental or PTEN-deficient HCT116 cells plus or minus irradiation (10 Gy). DAPI staining is included in the merged images to visualize nuclei. IR, ionizing radiation. Bars, 10 µm.

Article Snippet: Monoclonal antibodies against Chk1 (G-4) and actin (C-2) and polyclonal antibodies against Cdc2 phosphorylated at Y15 were purchased from Santa Cruz Biotechnology, Inc. Polyclonal antibodies against pS345 Chk1 and Mre11 were obtained from Cell Signaling Technology, whereas monoclonal antibody against α-tubulin (DM1A) was obtained from Sigma-Aldrich.

Techniques: Western Blot, Expressing, Irradiation, Staining

Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Antibodies against tubulin (TU-02; sc-8035), c-myc (9E10; sc-40), and p53 (DO-1; sc-126) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing FLAG epitope (#14793), BCAT1 (#88785), KU-80 (#2180), KU-70 (#4588), phosphorylated H2AX (pS139; #9718), phosphorylated DNA-PKcs (pS2056; #68716), total DNA-PKcs (#12311), phosphorylated ATM (pS1981; #5883), total ATM (#2873), phosphorylated CHK1 (pS345; #2348), phosphorylated CHK2 (pT68; #2197), total CHK2 (#6334), phosphorylated TP53 (pS15; #12571), and GADPH (#5174) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Antibodies against tubulin (TU-02; sc-8035), c-myc (9E10; sc-40), and p53 (DO-1; sc-126) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing FLAG epitope (#14793), BCAT1 (#88785), KU-80 (#2180), KU-70 (#4588), phosphorylated H2AX (pS139; #9718), phosphorylated DNA-PKcs (pS2056; #68716), total DNA-PKcs (#12311), phosphorylated ATM (pS1981; #5883), total ATM (#2873), phosphorylated CHK1 (pS345; #2348), phosphorylated CHK2 (pT68; #2197), total CHK2 (#6334), phosphorylated TP53 (pS15; #12571), and GADPH (#5174) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Antibodies against tubulin (TU-02; sc-8035), c-myc (9E10; sc-40), and p53 (DO-1; sc-126) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing FLAG epitope (#14793), BCAT1 (#88785), KU-80 (#2180), KU-70 (#4588), phosphorylated H2AX (pS139; #9718), phosphorylated DNA-PKcs (pS2056; #68716), total DNA-PKcs (#12311), phosphorylated ATM (pS1981; #5883), total ATM (#2873), phosphorylated CHK1 (pS345; #2348), phosphorylated CHK2 (pT68; #2197), total CHK2 (#6334), phosphorylated TP53 (pS15; #12571), and GADPH (#5174) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay