Journal: Oncogene

Article Title: Transgenic expression of E2F3a causes DNA damage leading to ATM-dependent apoptosis.

doi: 10.1038/onc.2008.138

Figure Lengend Snippet: Figure 2 Overexpression of E2F3a activates ATM. (a) Western blot analysis was performed on epidermal lysates from nontrans- genic (lanes 1,2, 5 and 6) or K5 E2F3a transgenic (lanes 3, 4, 7 and 8) mice that were either wild-type (lanes 1–4) or null (lanes 5–8) for Atm. Antibodies specific for E2F3, phospho-ATM S1981 and b-tubulin were used as indicated. (b) Skin sections from wild-type, K5 E2F3a, Atm/ and K5 E2F3a Atm/ mice were immunohis- tochemically stained for the phosphorylated form of p53 (serine 18 in mouse). Positively stained epidermal keratinocytes were identi- fied microscopically and the average number per 10 mm of linear epidermis from at least three mice per group is presented. (c) Western blot analysis was performed on lysates from primary NHFs (lanes 1–3) or primary fibroblasts from an AT patient (lanes 4–6) infected with AdCMV empty vector (lanes 1 and 4), treated with etoposide as a positive control for DNA damage (lanes 2 and 5) or infected with AdE2F3a (lanes 3 and 6). Antibodies specific for E2F3, phospho-p53 S15, phospho-ATM S1981 and b-tubulin were used as indicated. ATM, ataxia telangiectasia mutated; NHFs, normal human fibroblasts. *indicates statistical significance at Po0.05.

Article Snippet: The following antibodies were used to detect the indicated protein: phospho-ATM S1981 (Rockland, Gilbertsville, PA, USA), E2F3 (Santa Cruz Biotechnology, C-18), b-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, H-235), phospho-p53 S15 (Cell Signaling Technology), b-actin (Santa Cruz Biotechnology, H-2350).

Techniques: Over Expression, Western Blot, Transgenic Assay, Staining, Infection, Plasmid Preparation, Positive Control

Journal: Oncotarget

Article Title: Activation of ATR-Chk1 pathway facilitates EBV-mediated transformation of primary tonsillar B-cells

doi: 10.18632/oncotarget.14120

Figure Lengend Snippet: TBCs where inoculated with EBV-B95.8 (MOI 8). Non-inoculated TBCs at 24 hours post isolation were used as negative control (uninfected). Lymphoblastoid cell lines (LCLs) treated with Etoposide for 6 hours served as positive control. ( A ) Cells were harvested at the indicated time points, and expression of total ATM or Chk2 and pATM S1981 or pChk2 T68 was analyzed by western blotting. ( B ) ATM and Chk2 phosphorylation and total protein amount were quantified by densitometric analysis. Data are represented as ratio of phosphorylated-to-total. Quantification was performed using the software imageJ 1.49t. ( C ) Total KAP1 and pKAP1 S824 was analyzed by western blotting. The results shown are representative of 3 independent experiments. ( D ) ATM, Chk2 and KAP1 mRNA expression was determined in EBV-inoculated B-cells at the indicated time points by RTqPCR relative to 18 S RNA and shown as fold change relative to non-inoculated cells. Data are presented as mean ± SEM of 4 donors.

Article Snippet: Primary antibodies used in these studies included rabbit anti-pChk2 (Thr68) (clone C13C1; Cell Signaling), rabbit anti-Chk2 (Cell Signaling), rabbit anti-pATR (Ser428) (Cell Signaling), rabbit anti-ATR (Cell Signaling), rabbit anti-pChk1 (Ser345) (clone 133D3; Cell Signaling), rabbit anti β-actin (Cell Signaling), mouse anti-ATM (clone 2C1; GeneTex, Irvine, CA), rabbit anti-KAP1 (GeneTex), rabbit anti-pKAP1 (S824) (Bethyl, Montgomery, TX), rabbit anti-pATM (Ser1981) (Clone EP1890Y, Abcam, Cambridge, UK), mouse anti-Chk1 (clone G-4; Santa Cruz, Heidelberg, Germany).

Techniques: Isolation, Negative Control, Positive Control, Expressing, Western Blot, Software

Journal: Oncotarget

Article Title: Activation of ATR-Chk1 pathway facilitates EBV-mediated transformation of primary tonsillar B-cells

doi: 10.18632/oncotarget.14120

Figure Lengend Snippet: Peripheral B-cells were inoculated with EBV-B95.8 (MOI 8). Non-inoculated peripheral B-cells at 24 hours post isolation served as negative control (Uninfected), Lymphoblastoid cell lines (LCLs) derived from TMCs or PBMCs treated with Etoposide for 1 hours served as positive control. Detection of MRE11 served as loading control. ( A ) Cells were harvested at the indicated time points and expression of total ATR or Chk1 and pATR S428 or pChk1 S345 was analyzed by western blotting. ( B ) Cells were harvested at the indicated time points, and expression of total ATM or Chk2 and pATM S1981 or pChk2 T68 was analyzed by western blotting. The results shown are representative of 3 independent experiments. ( C ) ATR, ATM, Chk1 and Chk2 phosphorylation and total protein amount were quantified by densitometric analysis. Data are represented as ratio of phosphorylated-to-total. Quantification was performed using the software imageJ 1.49t. * = Not detectable.

Article Snippet: Primary antibodies used in these studies included rabbit anti-pChk2 (Thr68) (clone C13C1; Cell Signaling), rabbit anti-Chk2 (Cell Signaling), rabbit anti-pATR (Ser428) (Cell Signaling), rabbit anti-ATR (Cell Signaling), rabbit anti-pChk1 (Ser345) (clone 133D3; Cell Signaling), rabbit anti β-actin (Cell Signaling), mouse anti-ATM (clone 2C1; GeneTex, Irvine, CA), rabbit anti-KAP1 (GeneTex), rabbit anti-pKAP1 (S824) (Bethyl, Montgomery, TX), rabbit anti-pATM (Ser1981) (Clone EP1890Y, Abcam, Cambridge, UK), mouse anti-Chk1 (clone G-4; Santa Cruz, Heidelberg, Germany).

Techniques: Isolation, Negative Control, Derivative Assay, Positive Control, Expressing, Western Blot, Software

Journal: The Journal of Experimental Medicine

Article Title: ATM regulates ATR chromatin loading in response to DNA double-strand breaks

doi: 10.1084/jem.20051923

Figure Lengend Snippet: Analysis of ATM, Chk2, and Chk1 activation in response to DNA damage. (A) Western blot detection of ATM-1981 P , ATM, Chk2-T68 P , Chk2, Chk1-S345 P , and Chk1 in Raji human lymphoblastoid cells exposed to DNA-damaging conditions. Where indicated, ATM activity was inhibited by a previous 15-min treatment with 20 μM wortmannin (WM). (B) Evaluation of Chk1 phosphorylation dynamics in control and AT human cells in response to DNA damage. (C) Western blot analysis of ATR, Chk1-S345 P , Chk1, ATM-1981 P , and ATM levels in extracts from control (ATR wt ) and ATR hypomorphic (ATR Seckel ) human cells irradiated with 20 Gy (or mock irradiated [C]). β-actin levels were evaluated as a loading control. (D) Analysis of Chk1 phosphorylation in ATR flox/− cells 48 h after infection with Cre-expressing adenovirus (ATR Δ/− ) or mock infection (ATR flox/− ). (E) The same analysis as in D, in which the loading was normalized to eliminate differences in total Chk1 levels. In all cases, exponentially growing cells were irradiated with 20 Gy, and the extracts were performed 45 min after irradiation (IR) or 2 h after exposure to 2 mM hydroxyurea (HU).

Article Snippet: ATM-1981 P (Rockland Immunochemicals, Inc.), ATM (Novus Biologicals), ATR (Serotec), Chk2 (Upstate Biotechnology), Chk1 (Novocastra), β-actin (Sigma-Aldrich), Chk2-T68 P , and Chk1-S345 P (Cell Signaling Technology) primary antibodies were used in this study.

Techniques: Activation Assay, Western Blot, Activity Assay, Phospho-proteomics, Control, Irradiation, Infection, Expressing

Journal: The Journal of Experimental Medicine

Article Title: ATM regulates ATR chromatin loading in response to DNA double-strand breaks

doi: 10.1084/jem.20051923

Figure Lengend Snippet: Nuclear retention of ATM and ATR in response to DSBs. (A) The association of ATM and ATR with chromatin in the various treatments was evaluated in Raji cells by a serial fractionation protocol (see Materials and methods). Fraction I (cytoplasmic) and fraction III (chromatin bound) are shown from the same extracts. ATM-1981 P and Chk2-T68 P levels are shown as controls of the wortmannin inhibitory effect. No detectable Chk2-T68 P is bound to the chromatin with this protocol. The levels of the replication factor ORC2 are shown as a loading control in both cases because it is present both in the cytoplasmic as well as in a nuclear fraction. Notably, the association of ORC2 with chromatin does not change in response to DNA damage. The figure illustrates the common observation of a set of experiments ( n = 5). (B) Chromatin-bound fraction analysis in control and AT human cell lines. Treatments: C, control; IR, 20 Gy irradiation for 45 min; HU, 2 mM hydroxyurea for 2 h.

Article Snippet: ATM-1981 P (Rockland Immunochemicals, Inc.), ATM (Novus Biologicals), ATR (Serotec), Chk2 (Upstate Biotechnology), Chk1 (Novocastra), β-actin (Sigma-Aldrich), Chk2-T68 P , and Chk1-S345 P (Cell Signaling Technology) primary antibodies were used in this study.

Techniques: Fractionation, Control, Irradiation

Journal: The Journal of Experimental Medicine

Article Title: ATM regulates ATR chromatin loading in response to DNA double-strand breaks

doi: 10.1084/jem.20051923

Figure Lengend Snippet: Interactions between ATM, ATR, Chk2, and Chk1 in the DNA damage response. The diagram illustrates the molecular determinants that control the responses to IR and HU initiated in the different stages of the cell cycle. Whereas ATR and Chk1 activities in checkpoint signaling are restricted to cells beyond S phase, ATM activity is necessary throughout the cell cycle.

Article Snippet: ATM-1981 P (Rockland Immunochemicals, Inc.), ATM (Novus Biologicals), ATR (Serotec), Chk2 (Upstate Biotechnology), Chk1 (Novocastra), β-actin (Sigma-Aldrich), Chk2-T68 P , and Chk1-S345 P (Cell Signaling Technology) primary antibodies were used in this study.

Techniques: Control, Activity Assay