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: Cells were washed twice in staining media (PBS containing 1% BSA), resuspended in staining media containing Chk2-T68 P or Chk1-S345 P primary antibodies (Cell Signaling Technology), and incubated for 1 h at room temperature.

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: Flow cytometry analysis of Chk1 and Chk2 phosphorylation during the cell cycle. An aliquot of the same Raji cells that were used for Western blotting or chromatin fractionation was analyzed in parallel with multiparametric flow cytometry for DNA content with propidium iodide and for Chk1-S345 P (A and B) or Chk2-T68 P (C and D) levels with the corresponding specific antibodies (see Materials and methods). Note the distinct increase in Chk1 phosphorylation in irradiated G2 cells (red asterisk). Dashed lines are incorporated in each case to illustrate the phosphorylation dynamics. Doses used: 20 Gy IR; 2 mM HU, and 20 μM wortmannin. C, control; PI, propidium iodide.

Article Snippet: Cells were washed twice in staining media (PBS containing 1% BSA), resuspended in staining media containing Chk2-T68 P or Chk1-S345 P primary antibodies (Cell Signaling Technology), and incubated for 1 h at room temperature.

Techniques: Flow Cytometry, Phospho-proteomics, Western Blot, Fractionation, Irradiation, Control

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: Cells were washed twice in staining media (PBS containing 1% BSA), resuspended in staining media containing Chk2-T68 P or Chk1-S345 P primary antibodies (Cell Signaling Technology), and incubated for 1 h at room temperature.

Techniques: Control, Activity Assay

Journal:

Article Title: The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1

doi: 10.1101/gad.1547007

Figure Lengend Snippet: Rad9 Ser387 is involved in Chk1 phosphorylation. (A) Constructs used in this study. Rad9 and TopBP1 are not drawn to scale. Rad9 PCNA-like N-terminal domain and tail are indicated. P in tail indicates intact phosphorylation sites, whereas A indicates phosphorylation sites mutated to Ala. BRCT domains in TopBP1 are indicated; AD is the activation domain. (B) Rad9−/− DT40 cells were transiently transfected with empty vector (EV) and vectors encoding untagged wild-type Rad9 (WT); Rad9-9A (9A), the mutant lacking nine C-terminal phosphorylation sites; and Rad9-9A to which the indicated phosphorylation sites were restored (denoted as Rad9-9A + site). Following treatment with 10 mM HU for 1 h, transfected Rad9−/− DT40 cells and parental (wild-type) DT40 cells were lysed, separated by SDS-PAGE, and sequentially immunoblotted for phospho-Ser345-Chk1, Chk1, and Rad9. The multiple bands present in the Rad9 immunoblots are due to various forms of phosphorylated Rad9 (Volkmer and Karnitz 1999). The dotted line indicates the juxtaposition of nonadjacent regions of the same gel. (C) Lysates from HEK293 cells transiently transfected with empty vector (EV) or vectors encoding S-tagged wild-type Rad9 (WT), Rad-9A (9A), or the indicated Rad9-9A add-back expression plasmids were precipitated with S-protein agarose beads. Bound proteins were sequentially immunoblotted for endogenous TopBP1 (top) and Rad9 (middle). The multiple bands present in the Rad9 immunoblots are due to various forms of phosphorylated Rad9 (Volkmer and Karnitz 1999). (Bottom) A portion of the lysate was also immunoblotted to demonstrate equal TopBP1 levels in all samples.

Article Snippet: Lysates were separated by SDS-PAGE (10% gel), transferred to Immobilon-P (Millipore), and immunoblotted, as indicated, with a rabbit monoclonal antibody recognizing P-Ser 345 -Chk1 (133D3; Cell Signaling Technology); mouse monoclonal antibodies recognizing S-peptide ( Hackbarth et al. 2004 ), Chk1 (G-4; Santa Cruz Biotechnology), or PCNA (PC10; Santa Cruz Biotechnology); or rabbit polyclonal antisera recognizing Rad17 ( Volkmer and Karnitz 1999 ), Rad9 ( Volkmer and Karnitz 1999 ), or TopBP1 (BL893; Bethyl Laboratories).

Techniques: Phospho-proteomics, Construct, Activation Assay, Transfection, Plasmid Preparation, Mutagenesis, SDS Page, Western Blot, Expressing

Journal:

Article Title: The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1

doi: 10.1101/gad.1547007

Figure Lengend Snippet: Rad9 activates Chk1 by binding TopBP1. Rad9−/− (A,B) or Rad17−/− (C–E) DT40 cells were transiently transfected with the indicated plasmids and treated with 10 mM HU for 1 h, and lysates were separated by SDS-PAGE and sequentially immunoblotted to detect phospho-Ser345-Chk1 and Chk1. To detect fusion protein and Rad17 expression, the samples were immunoblotted to detect S-tagged fusions (A–C,E), Rad17 (D), or PCNA (D). Transfections were with empty vector (EV); vectors expressing S-tagged wild-type Rad9 (WT), Rad9-9A (9A), Rad9-9A fused to full-length TopBP1 (9A–TopBP1), Rad9-9A–TopBP1 in which the WDDP motif in the AD was deleted from TopBP1 (9A–TopBP1–ΔWDDP), Rad9-9A fused to the TopBP1 AD (9A–AD), a tailless Rad9 fused to the AD (Δtail–AD), Rad17, or H2B fused to the TopBP1–AD (H2B–AD); or a vector expressing PCNA fused to the Rad9 tail (PCNA–Rad9 tail) or the TopBP1 AD (PCNA–AD). The multiple bands present in the Rad9 immunoblots are due to various forms of phosphorylated Rad9 (Volkmer and Karnitz 1999). Asterisk indicates nonspecific immunoreactive bands (A) or putative degradation products of the PCNA fusion proteins (D).

Article Snippet: Lysates were separated by SDS-PAGE (10% gel), transferred to Immobilon-P (Millipore), and immunoblotted, as indicated, with a rabbit monoclonal antibody recognizing P-Ser 345 -Chk1 (133D3; Cell Signaling Technology); mouse monoclonal antibodies recognizing S-peptide ( Hackbarth et al. 2004 ), Chk1 (G-4; Santa Cruz Biotechnology), or PCNA (PC10; Santa Cruz Biotechnology); or rabbit polyclonal antisera recognizing Rad17 ( Volkmer and Karnitz 1999 ), Rad9 ( Volkmer and Karnitz 1999 ), or TopBP1 (BL893; Bethyl Laboratories).

Techniques: Binding Assay, Transfection, SDS Page, Expressing, Plasmid Preparation, Western Blot

Journal:

Article Title: The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1

doi: 10.1101/gad.1547007

Figure Lengend Snippet: Chk1 activation correlates with cell survival. Rad9−/− (A) or Rad17−/− (B) DT40 cells were cotransfected with the indicated plasmids (described in Fig. 3) and pEGFP-N1. The following day, EGFP-positive cells were purified by fluorescence-activated cell sorting, treated with HU, and stained with Hoechst 33258. For each sample, 250 cells were examined by microscopy. Apoptotic cells were identified based on nuclear morphology. Error bars indicate standard deviation of three to four independent experiments. (C) Model of Rad9’s role in Chk1 activation. See the text for details.

Article Snippet: Lysates were separated by SDS-PAGE (10% gel), transferred to Immobilon-P (Millipore), and immunoblotted, as indicated, with a rabbit monoclonal antibody recognizing P-Ser 345 -Chk1 (133D3; Cell Signaling Technology); mouse monoclonal antibodies recognizing S-peptide ( Hackbarth et al. 2004 ), Chk1 (G-4; Santa Cruz Biotechnology), or PCNA (PC10; Santa Cruz Biotechnology); or rabbit polyclonal antisera recognizing Rad17 ( Volkmer and Karnitz 1999 ), Rad9 ( Volkmer and Karnitz 1999 ), or TopBP1 (BL893; Bethyl Laboratories).

Techniques: Activation Assay, Purification, Fluorescence, FACS, Staining, Microscopy, Standard Deviation

Journal: Oncotarget

Article Title: Lysophosphatidylcholine induces cytotoxicity/apoptosis and IL-8 production of human endothelial cells: Related mechanisms

doi: 10.18632/oncotarget.22425

Figure Lengend Snippet: EAHY endothelial cells were exposed to different concentrations of LPC. Immunofluorescent (IF) microscopic observation was utilized to determine the expression of ( A ) p-ATM, ( B ) p-ATR, ( C ) p-Chk1, and ( D ) p-Chk2 in endothelial cells. One representative IF picture was shown. (blue – DAPI, green – p-ATM, red – p-ATR, p-Chk1, or p-Chk2).

Article Snippet: Antibodies against cdc2 (sc-54), cyclin B1 (sc-245), p-ATM (Ser1981, sc-47739), p-ATR (Ser428, sc-109912), p-Chk1 (Ser345, sc-17922), p-Chk2 (Thr68, sc-16297-R), p-Akt 1/2/3 (Ser473, sc-514032) and glyceraldehtde 3-phosphate dehydrogenase (GAPDH) (sc-32233) were obtained from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Expressing