Journal: The Journal of Cell Biology

Article Title: The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks

doi: 10.1083/jcb.201205059

Figure Lengend Snippet: DNA damage signaling is not affected by p400 knockdown. (A and B) U2OS cells transfected with control or p400 siRNA were irradiated (8 Gy) or not 48 h later and subjected to FK2 immunofluorescence. Representative images (A) and quantifications are shown (B). Bar, 10 µm. Results are the mean ± SD from three independent experiments. (C and F) Automatized analysis of ubiquitin (FK2; C) or 53BP1 (F) foci in U2OS cells exposed to 8 Gy. Note that in other experiments the decrease was less pronounced after the 30-min peak of FK2 foci. (D and G) Automatized analysis of ubiquitin (FK2; D) or 53BP1 (G) foci in U2OS cells exposed to 2 Gy. (E and H) Automatized analysis of ubiquitin (FK2; E) or 53BP1 (H) foci in 293T cells exposed to 8 Gy. For C–H, the data shown are from a single representative experiment ( n = 200) out of three repeats.

Article Snippet: The commercial p400 antibody was purchased from Abcam, the Rad51 antibodies from Santa Cruz Biotechnology, Inc. (Rad51-H92; for ChIPs and some Western blot experiments) or from EMD Millipore (for some Western blot experiments), the p21 antibody (C19) from Santa Cruz Biotechnology, Inc., the HA antibody from Covance (HA-11), the myc antibody from Roche (9E10), the γH2AX antibodies from EMD Millipore for immunofluorescence experiments (JBW301) and from Epitomics for Western blot experiments, the actin and γ-tubulin antibodies from Sigma-Aldrich (T6199), the phospho-ATM antibody from Cell Signaling technology (10H11.E12), the lamin A/C antibody from Tebu-Bio (636), the phospho-RPA antibodies from Bethyl Laboratories, Inc. (S4-8) for immunofluorescence and (S33) for Western blot experiments, p-ChK2 antibody from Cell Signaling Technology (T68), BRCA1 antibody from Santa Cruz Biotechnology, Inc. (D-9), the FK2 antibody from EMD Millipore, the Rad52 antibody from Abcam, and the 53BP1 antibody from Novus Biologicals.

Techniques: Knockdown, Transfection, Control, Irradiation, Immunofluorescence, Ubiquitin Proteomics

Journal: Nucleic Acids Research

Article Title: The COP9 signalosome is vital for timely repair of DNA double-strand breaks

doi: 10.1093/nar/gkv270

Figure Lengend Snippet: Elimination of the Atm phosphorylation site on murine endogenous Csn3 impairs the DSB response in mice. ( A ) Endogenous Csn3 is not phosphorylated on S410 in response to DSBs in various tissues of mice expressing a mutated protein with the S410A amino acid substitution. Ten-day-old mice were irradiated with 10 Gy of IR, and 1 h later tissues were collected and processed for immunoblotting analysis with the indicated antibodies. Phosphorylation of the Atm substrate Kap-1 on S824 served to monitor ATM activity. ( B ) Kaplan–Meier survival curve of 1-month old mice with various genotypes in the Cops3 locus encoding Csn3 following whole body irradiation with 6.5 Gy of IR. 10–20 animals of each genotype were irradiated. ( C ) Clonogenic survival of MEFs with different genotypes after treatment with various NCS doses. Cells expressing non-phosphorylatable Csn3 exhibit intermediate radiomimetic sensitivity between that of wild-type and that of Atm-deficient cells. ( D and E ). Lack of an Atm phosphorylation site on Csn3 slows the disappearance of DNA damage-induced γH2AX and 53BP1 nuclear foci. MEFs with the indicated version of endogenous Csn3 were irradiated with 1 Gy of IR, and subsequently fixed and stained with antibodies against γH2AX or 53BP1. Nuclear foci were counted in 150–250 cells per time point. ( F ) Similar analysis as in (D) and (E) using an antibody against Rad51. The diagram presents the fraction of cells which contained more than 15 nuclear foci. Experiments were carried out in triplicate, and one set of results is shown. Error bars represent SEM. Statistical analysis was based on Student's t test for γH2AX, 53BP1 and χ 2 for Rad51.

Article Snippet: Anti-53BP1 mouse monoclonal antibody was a generous gift from T. Halazonetis, anti-53BP1 polyclonal antibody was obtained from Novus Biologicals, LLC (Littleton CO, USA), anti-pS139-H2AX (γH2AX) polyclonal antibody and polyclonal antibodies against CSN subunits 1, 3 and 5 were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, USA), anti-RNF168, anti-H2A, anti-pS139-H2AX monoclonal antibody and anti-BRCA1 antibodies were obtained from Merck Millipore (Darmstadt, Germany), anti-H4 was obtained from Abcam (Cambridge, MA, USA), anti-HSC70 monoclonal antibody was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and anti-Cullin 1 antibody was obtained from Invitrogen Corporation (Camarillo, CA, USA).

Techniques: Phospho-proteomics, Expressing, Irradiation, Western Blot, Activity Assay, Staining