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:

Article Title: ATM Activation and Its Recruitment to Damaged DNA Require Binding to the C Terminus of Nbs1

doi: 10.1128/MCB.25.13.5363-5379.2005

Figure Lengend Snippet: Recruitment of ATM to DSBs precedes its autophosphorylation, and ATM autophosphorylation in Nbs1-depleted extracts can be restored by the C-terminal 147 amino acids of Nbs1. (A) The anti-C147 antibodies prevent ATM binding to the linear plasmid DNA. Extracts preincubated with 300 ng/μl of control IgG or affinity-purified anti-C147 were incubated with magnetic avidin beads coupled with a one-end-biotinylated 2-kb DNA fragment derived from pBluescript. Extracts were incubated with extracts at room temperature for 5 min. Proteins associated with beads were immunoblotted for S1981-ATM (unphosphorylated ATM), pS1981-ATM, total ATM, Ku70, and NBS1. (B) S1981 phosphorylation is not a prerequisite for the interaction of ATM with linear plasmid DNA. Reactions were similar to panel A except samples were not preincubated with antibodies and samples were taken at 5-min intervals following addition of DNA beads. (C) The anti-C147 antibodies prevent ATM binding to damaged sperm chromatin. Extracts preincubated with 250 ng/μl of control IgG or affinity-purified anti-C147 were incubated with 2,500 EcoRI-treated demembranated sperm nuclei/μl. Extracts were incubated at room temperature for 5 min. Proteins associated with chromatin were immunoblotted for S1981-ATM (unphosphorylated ATM), pS1981-ATM, total ATM, NBS1, and Ku70. (D) A recombinant fragment of Nbs1 that contains the Mre11- and ATM-binding domains restores ATM autophosphorylation to an Nbs1-depleted extract. Anti-C147 antibodies were used to deplete Nbs1 from a Xenopus egg extract. Buffer, 100 ng/μl GST, GST-C147, GST-C97, or GST-C50 was added to the extract together with linearized plasmid DNA. After 15 min of incubation, extracts were then immunoblotted for pS1981-ATM and total ATM. (E) Linearized plasmid DNA and damaged sperm chromatin induce ATM autophosphorylation in an Nbs1-depleted extract supplemented with a recombinant fragment of Nbs1 that contains the Mre11- and ATM-binding domains. GST-C147 (100 ng/μl) was incubated with buffer, 1 ng/μl of linearized pBluescript, or 2,500 sperm chromatin treated with EcoRI/μl. After 15 min of incubation, extracts were then immunoblotted for pS1981-ATM and total ATM. (F) The C147 fragment of Nbs1 restores ATM binding to linearized plasmid DNA and damaged sperm chromatin in an Nbs1-depleted extract. The Nbs1-depleted extract supplemented with 100 ng/μl of GST or GST-C147 was incubated with magnetic avidin beads coupled with one-end-biotinylated 2-kb fragments derived from pBluescript or sperm chromatin treated with EcoRI. After 2 min of incubation, DNA beads and chromatin were isolated and proteins associated with DNA beads or damaged chromatin were immunoblotted for S1981-ATM, pS1981-ATM, total ATM, and Ku70. Similar results were observed after a 10-min incubation except that the S1981-ATM signal was very weak (Z. You and T. Hunter, unpublished data).

Article Snippet: The pS1981-ATM and S1981-ATM antibodies were purchased from Rockland Immunochemicals (catalog no. 600-401-400 and 600-401-398).

Techniques: Binding Assay, Plasmid Preparation, Affinity Purification, Incubation, Avidin-Biotin Assay, Derivative Assay, Recombinant, Isolation

Journal:

Article Title: A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies

doi: 10.1261/rna.2340405

Figure Lengend Snippet: eIF4E and eIF4E-T colocalize with LSm1 in distinct cytoplasmic foci. HeLa SS6 cells were grown on coverslips, fixed, and stained with antibodies specific for eIF4E (monoclonal anti-eIF4E, Santa Cruz Biotechnology) (A,N), LSm1 (B,E,H,K,Q,T), eIF4G (G,J,M), or eIF4E-T (P). Alternatively, cells were transfected with plasmids encoding YFP-eIF4E (D) or YFP-eIF4E-T (S). Panels C,F,I,L,O,R,U show the merged picture of the proceeding two panels, with overlaying signals appearing yellow. In panels J–O, cells were treated with 100 μM arsenite for 45 min at 37°C to induce stress granules (indicated by arrows). Cells were analyzed by confocal fluorescence microscopy. Scale bars = 10 μm.

Article Snippet: The following primary antibodies were used in this study: affinity-purified anti-LSm1 polyclonal peptide antibodies (1:500 dilution) (see Ingelfinger et al. 2002 ); rabbit anti-Xp54 (gift from J. Sommerville, 1:200); rabbit anti-Ccr4 (gift from E. Wahle, 1:100); rabbit anti-eIF4E serum ( Naegele and Morley 2004 ), goat anti-eIF4G (1:400); mouse anti-eIF4E (P-2, 1:800); and goat anti-eIF4E-T (E-18, 1:400) (purchased from Santa Cruz Biotechnology).

Techniques: Staining, Transfection, Fluorescence, Microscopy