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

Journal: BMC Cancer

Article Title: SC-III3, a novel scopoletin derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation

doi: 10.1186/1471-2407-14-987

Figure Lengend Snippet: Effect of SC-III3 on DNA damage and ATM/ATR pathways. (A) HepG2 cells were treated with SC-III3 for 24 hours. Western blot analysis of p-H2AX, total H2AX, p-Chk1 (Ser280) and p-Chk1 (Ser296) response to SC-III3. (B) HepG2 cells were treated with SC-III3 for 24 hours. Western blot analyses of the ATM/ATR signaling related proteins in HepG2 cells. (C) HepG2 cells were treated with 1 μM SC-III3 for different times as indicated. Western blot analyses of p-ATM, p-ATR, p-Chk1, p-Chk2, Cdc25C, p-CDK2, p53, and p21 in HepG2 cells. These experiments were done in triplicates.

Article Snippet: Antibodies against p-ATM (Ser1981), ATM, p-ATR (Ser428), ATR, p-Chk1 (Ser345), p-Chk1 (Ser280), p-Chk1 (Ser296), Chk1, p-Cdk2 (Tyr15), p53, p21 were purchased from Cell Signaling Technology (Danvers, MA). p-Chk2 (Thr68), Chk2, Cdc25A, p-H2AX(Ser139), H2AX antibodies were purchased from EnoGene Biotech (Nanjing, China), GAPDH monoclonal antibodies were purchased from Kangchen Bio-tech (Shanghai, China); Cdk2, cyclinA, cyclinE, cyclinB, Bax, Bcl-2 monoclonal antibodies were purchased from Bioworld (Georgia, USA).

Techniques: Western Blot

Journal: BMC Cancer

Article Title: SC-III3, a novel scopoletin derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation

doi: 10.1186/1471-2407-14-987

Figure Lengend Snippet: Role of ATM/Chk1/Chk2 in SC-III3-mediated S cell cycle arrest. (A, B) HepG2 cells were exposed to 1 μM SC-III3 for 24 h with or without adding of 10 μM Ku55933 or 0.3 μM UCN-01. Then, cells were analyzed for cell cycle distribution by flow cytometry. Cells in G0/G1, S, and G2/M phases were quantified and presented. (C) HepG2 cells were exposed to 1 μM SC-III3 with or without adding of 10 μM Ku55933 for 24 h. Then, western blot analyses of p-ATM, p-ATR, p-Chk1, p-Chk2, Cdc25C, p-CDK2, p53, and p21 in HepG2 cells. (D) HepG2 cells were exposed to 1 μM SC-III3 with or without adding of 0.3 μM UCN-01 for 24 h. Then, western blot analyses of p-Chk1, p-Chk2, p-Cdk2, and Cdc25A in HepG2 cells. The Data were presented as mean ± SEM for three separate experiments. The difference were significant at ** p < 0.01 compared with SC-III3 (1 μM) and ## p < 0.01 compared with SC-III3 (0 μM).

Article Snippet: Antibodies against p-ATM (Ser1981), ATM, p-ATR (Ser428), ATR, p-Chk1 (Ser345), p-Chk1 (Ser280), p-Chk1 (Ser296), Chk1, p-Cdk2 (Tyr15), p53, p21 were purchased from Cell Signaling Technology (Danvers, MA). p-Chk2 (Thr68), Chk2, Cdc25A, p-H2AX(Ser139), H2AX antibodies were purchased from EnoGene Biotech (Nanjing, China), GAPDH monoclonal antibodies were purchased from Kangchen Bio-tech (Shanghai, China); Cdk2, cyclinA, cyclinE, cyclinB, Bax, Bcl-2 monoclonal antibodies were purchased from Bioworld (Georgia, USA).

Techniques: Flow Cytometry, Western Blot