Journal: The Journal of Biological Chemistry

Article Title: The ATM Cofactor ATMIN Protects against Oxidative Stress and Accumulation of DNA Damage in the Aging Brain *

doi: 10.1074/jbc.M110.145896

Figure Lengend Snippet: ATMINΔ/Δ fibroblasts are highly susceptible to oxidative damage and exhibit aberrant DNA damage signaling. A, ATMIN+/+ and ATMINΔ/Δ fibroblasts were cultured at 3 or 20% oxygen for 1 passage (P1) or up to 4 passages (P4). Cells were fixed and stained for senescence-associated β-galactosidase. The histogram shows quantification of staining. B, ATMIN+/+ and ATMINΔ/Δ fibroblasts were treated as in A for 48 h following which cells were harvested and proteins were blotted for p21. C, ATMIN+/+ and ATMINΔΔ fibroblasts were treated as in A for 4 passages either in the absence or presence of 100 μm NAC. Cells were fixed and stained with senescence-associated β-galactosidase and the percentage of positive cells was counted. The histogram shows quantification from two experiments. D, ATMIN+/+ and ATMINΔΔ fibroblasts were cultured at 20% oxygen for 4 passages followed by fixation with 4% PFA and staining with pS139-γH2aX and pS1987-ATM. Colocalizing and noncolocalizing foci are indicated by yellow and white arrowheads, respectively. The histogram shows quantification of the percentage of pS139-γH2aX-positive cells that do not have colocalizing pS1987-ATM foci. E, ATMIN-deficient fibroblasts display reduced DNA damage signaling in response to treatment with paraquat. Cells were treated with 10 μm paraquat for 10 h and cell extracts were blotted for pS1987-ATM, ATM, pS824-Kap1, Kap1, pS15-P53, P53, pT68-Chk2, Chk2, and β-actin.

Article Snippet: The following antibodies were used: pS1981-ATM (10H11.E12 Cell signaling), ATM (2C1, Santa Cruz), p53 (DO-1, Santa Cruz), pS15-p53 (Cell signaling), P21 (C19, Santa Cruz), pS824-Kap1 (Bethyl Laboratories), Kap1 (Bethyl Laboratories), β-actin (A5060, Sigma), pS957-SMC1 (5D11G5, Millipore), SMC1 (AB3908, Millipore), pT68-Chk2 (Cell Signaling), Chk2 (clone 7, Millipore), and HRP-conjugated goat anti-mouse/rabbit IgG (Sigma).

Techniques: Cell Culture, Staining

Journal: The Journal of Biological Chemistry

Article Title: The ATM Cofactor ATMIN Protects against Oxidative Stress and Accumulation of DNA Damage in the Aging Brain *

doi: 10.1074/jbc.M110.145896

Figure Lengend Snippet: Quantification of extent of ATM signaling in response to paraquat treatment

Article Snippet: The following antibodies were used: pS1981-ATM (10H11.E12 Cell signaling), ATM (2C1, Santa Cruz), p53 (DO-1, Santa Cruz), pS15-p53 (Cell signaling), P21 (C19, Santa Cruz), pS824-Kap1 (Bethyl Laboratories), Kap1 (Bethyl Laboratories), β-actin (A5060, Sigma), pS957-SMC1 (5D11G5, Millipore), SMC1 (AB3908, Millipore), pT68-Chk2 (Cell Signaling), Chk2 (clone 7, Millipore), and HRP-conjugated goat anti-mouse/rabbit IgG (Sigma).

Techniques:

Journal: Nature Communications

Article Title: RNAPII-dependent ATM signaling at collisions with replication forks

doi: 10.1038/s41467-023-40924-4

Figure Lengend Snippet: Antibodies used in this study

Article Snippet: pS824-KAP1 (BL-246-7B5) , ab243870 , Abcam , H M , IP, IHC-P, WB, ICC/IF.

Techniques: Immunohistochemistry-IF, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Simple Western, Dot Blot

Journal: The EMBO Journal

Article Title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

doi: 10.1038/emboj.2012.86

Figure Lengend Snippet: KAP-1 phosphorylation on S824 is regulated by a PP4C–R3β complex. (A) In unperturbed cells, phosphorylation of KAP-1 on S824 is elevated in PP4-depleted cells. HeLa cells transfected with siRNAs against indicated PP4 subunits were harvested after 72 h and immunoblotting was performed with the indicated antibodies. (B) HeLa cells transfected with siRNAs against PP4C or PP4R3β were irradiated and harvested at the indicated times and p-KAP-1 was assessed by immunoblot using phospho-KAP-1 antibody (phosphoSerine 824). The kinetics of pS824-KAP-1 formation was monitored after irradiation by loading 20 μg of cell lysate per lane. (C) PP4R3β depletion attenuates pS824-KAP-1 turnover after IR. Primary human fibroblasts were transfected with control or PP4R3β siRNAs. After 72 h, cells were irradiated, fixed at the indicated times and immunostained for pS824-KAP1 (red), γH2AX (green) and DAPI (blue). Right Panel: The average pS824-KAP-1 signal intensity per nucleus was quantified using ImageJ software. Data represent average and s.d. of three independent experiments. (D) PP4C/PP4R3β interacts with KAP-1. HeLa S3 cells stably expressing empty vector (FH-Ctrl), FH-tagged KAP-1 (top and bottom panel), and PP4C or PP4R3β (middle panel) were subjected to immunoprecipitation using anti-FLAG beads at indicated time points before or after IR. PP4R3β or control siRNAs were transfected in cells 72 h prior to immunoprecipitation (right panel, bottom). The immunoprecipitate was probed with antibodies against endogenous KAP-1, PP4C and PP4R3β as indicated. Figure source data can be found with the Supplementary data.

Article Snippet: Identical results were achieved using commercial anti-KAP-1 pS824 from Bethyl, USA.

Techniques: Phospho-proteomics, Transfection, Western Blot, Irradiation, Control, Software, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

doi: 10.1038/emboj.2012.86

Figure Lengend Snippet: Hyperphosphorylation of S824-KAP-1 in PP4-silenced cells is not due to ectopic activation of ATM. (A) pS824-KAP-1 is lost rapidly in the absence of ongoing ATM activity. (Upper panel) 1BR3 primary fibroblasts were irradiated with 3-Gy IR and, 0.5 h later, were incubated with either DMSO or 10 μM of ATMi and harvested at the indicated times. Cells were fixed and immunostained for pS824-KAP-1 (red), γH2AX (green) and DAPI (blue). (Lowerpanel) GM02188 lymphoblastoid cells were irradiated with 10-Gy IR and, 20 min later, were incubated with DMSO or ATMi. Cells were harvested at the indicated times, processed and immunoblotted for pS824-KAP-1 and total KAP-1 (top panel). Immunoblot signal was quantified and plotted (lower panel). The half-lives of signal decay were calculated. (B) PP4C/PP4R3β depletion attenuates pS824-KAP-1 turnover after IR independent of ATM activity. HeLa cells were transfected with siRNAs. After 72 h, cells were irradiated with 10-Gy IR. At 0.5 h post IR, DMSO or ATMi was added. Cells were harvested at the indicated times and immunoblotting was performed and band intensities quantified using the Odyssey Infrared Imaging System. The intensity of pS824-KAP-1 was normalized relative to total KAP-1 and represented graphically. (C) PP4R3β depletion attenuates pS824-KAP-1 turnover after IR independent of ATM activity. Primary human fibroblasts were transfected with siRNAs. After 72 h, cells were irradiated with 3-Gy IR. At 0.5 h post IR, DMSO or ATMi was added. Cells were fixed at the indicated times and immunostained for pS824-KAP1 (red), γH2AX (green) and DAPI (not shown). The data were quantified as in Figure 2C. (D) PP4R3β regulates pS824-KAP-1 at late-repairing IRIF. Primary human fibroblasts were transfected with control or PP4R3β siRNA. After 72 h, cells were irradiated with 8-Gy IR. After 24 h, ATMi was added (t=0 min) and cells were fixed at the indicated times up to 120 min. Fixed cells were then immunostained for pS824-KAP-1 (red), γH2AX (green) and DAPI (blue). The average pS824KAP-1 signal intensity (from (A)) overlapping with γH2AX foci (i.e., ‘at IRIF’) was quantified using ImageJ software. Data represent average and s.d. of three independent experiments. The approximate half-life (t1/2) of pS824-KAP-1 signal following the addition of ATMi is indicated. Data represent average and s.d. of three independent experiments. Figure source data can be found with the Supplementary data.

Article Snippet: Identical results were achieved using commercial anti-KAP-1 pS824 from Bethyl, USA.

Techniques: Activation Assay, Activity Assay, Irradiation, Incubation, Western Blot, Transfection, Imaging, Control, Software

Journal: BMC Cancer

Article Title: Functional deficiency of NBN, the Nijmegen breakage syndrome protein, in a p.R215W mutant breast cancer cell line

doi: 10.1186/1471-2407-14-434

Figure Lengend Snippet: Assessment of cellular radiosensitivity in the colony formation assay. A . Cellular radiosensitivity of p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by the colony formation assay after irradiation at doses of 0, 0.1, 0.25, 0.5 or 1Gy. The surviving fraction is presented as the mean value with SEM from at least 3 independent experiments. B . Induction of PARP1 cleavage after irradiation with 2 Gy in p.R215W mutant cells (HCC1395) compared with wild type breast epithelial cells (MCF10A) as measured by immunoblotting of cleaved PARP1 (89 kDa) and total PARP1 (116 kDa). C. Immunoblot analysis of radiation-induced ATM signalling in HCC1395 cells compared with MCF10A. Cells were untreated or irradiated with 0.5, 1, 2, 4 or 6 Gy as indicated. Protein extracts were prepared 30 min after irradiation and were analysed through Western blotting for their immunoreactivity towards the phosphorylated forms of SMC1 (pSer966, top panel), KAP1 (p824, middle panel), and CHEK2 (pSer19, bottom panel). β-actin served as the loading control in each experiment.

Article Snippet: Antibodies to NBN, SMC1-pS966, KAP1-pS824 were obtained from Novus Biologicals (rabbit polyclonal); anti CHEK2-pS19 from Cell Signaling (rabbit polyclonal); anti RAD50 (mouse monoclonal) from Abcam; anti MRE11 (mouse monoclonal 12D7) from GeneTex; PARP1 and cleaved PARP1 (rabbit polyclonal) from Cell Signaling, and anti β-Actin (mouse monoclonal) from Sigma.

Techniques: Colony Assay, Mutagenesis, Irradiation, Western Blot, Control