Human/Mouse/Rat Phospho-ATM (S1981) Antibody

Detection of Human/Mouse/Rat Phospho-ATM (S1981) by Western Blot. Western blot shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with 1 µM camptothecin (CPT) for 1 hour. PVDF membrane was probed with 1 µg/mL Rabbit Anti-Human/Mouse/Rat Phospho-ATM (S1981) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1655) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (Catalog # HAF008). A specific band for Phospho-ATM (S1981) was detected at approximately 370 kDa (as indicated). The phospho-specificity of this antibody was supported by decreased labeling following treatment with 600 U lambda-phosphatase (lambda-PPase) for 1 hour. This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Detection of Human Phospho-ATM (S1981) by Simple Western^TM. Simple Western lane view shows lysates of HeLa human cervical epithelial carcinoma cell line untreated (-) or treated (+) with 1 µM Camptothecin (CPT) for 1 hour, loaded at 0.2 mg/mL. A specific band was detected for Phospho-ATM (S1981) at approximately 270 kDa (as indicated) using 10 µg/mL of Rabbit Anti-Human/Mouse/Rat Phospho-ATM (S1981) Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1655). This experiment was conducted under reducing conditions and using the 66-440 kDa separation system.

Detection of Human ATM by Western Blot Resveratrol activates ATM in transformed human cell lines.(a, b) Human HEK293T cells were treated with 0.01 mM ATM inhibitor (KU-55933) or vehicle (DMSO) for 1 hour, followed by incubation in media also containing 0.1 mM resveratrol (or DMSO), as indicated. After 30 minutes incubation with resveratrol, H2O2 (0.1 mM) or bleomycin (1 µg/ml) was added as indicated for 30 minutes before harvesting. Western blotting was performed with with antibodies directed against ATM, phospho-ATM Ser-1981, p53, and phospho-p53 Ser-15 as indicated. (c, d) Human HEK293T cells were treated with resveratrol, bleomycin, or both as in (a) and probed for gamma -H2AX foci by immunofluorescence. Cell images (291, 267, 216, and 260 cells, respectively) were analyzed for foci using Image J software, and the average number of foci per cell as well as the percentage of cells containing >5 foci were quantitated. (e, f) Human HCT116 cells were incubated with KU-55933, resveratrol, H2O2, and bleomycin as in (a, b) but additional phosphorylation targets were examined using antibodies directed against Smc1, phospho-Smc1 Ser-957, Kap1, phospho-Kap1 Ser-824, Nbs1, phospho-Nbs1 Ser-343, Chk2, and phospho-Chk2 Thr-68 as indicated. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0097969), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human ATM by Western Blot Resveratrol activates ATM in transformed human cell lines.(a, b) Human HEK293T cells were treated with 0.01 mM ATM inhibitor (KU-55933) or vehicle (DMSO) for 1 hour, followed by incubation in media also containing 0.1 mM resveratrol (or DMSO), as indicated. After 30 minutes incubation with resveratrol, H2O2 (0.1 mM) or bleomycin (1 µg/ml) was added as indicated for 30 minutes before harvesting. Western blotting was performed with with antibodies directed against ATM, phospho-ATM Ser-1981, p53, and phospho-p53 Ser-15 as indicated. (c, d) Human HEK293T cells were treated with resveratrol, bleomycin, or both as in (a) and probed for gamma -H2AX foci by immunofluorescence. Cell images (291, 267, 216, and 260 cells, respectively) were analyzed for foci using Image J software, and the average number of foci per cell as well as the percentage of cells containing >5 foci were quantitated. (e, f) Human HCT116 cells were incubated with KU-55933, resveratrol, H2O2, and bleomycin as in (a, b) but additional phosphorylation targets were examined using antibodies directed against Smc1, phospho-Smc1 Ser-957, Kap1, phospho-Kap1 Ser-824, Nbs1, phospho-Nbs1 Ser-343, Chk2, and phospho-Chk2 Thr-68 as indicated. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0097969), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human ATM by Western Blot Resveratrol activates ATM in transformed human cell lines.(a, b) Human HEK293T cells were treated with 0.01 mM ATM inhibitor (KU-55933) or vehicle (DMSO) for 1 hour, followed by incubation in media also containing 0.1 mM resveratrol (or DMSO), as indicated. After 30 minutes incubation with resveratrol, H2O2 (0.1 mM) or bleomycin (1 µg/ml) was added as indicated for 30 minutes before harvesting. Western blotting was performed with with antibodies directed against ATM, phospho-ATM Ser-1981, p53, and phospho-p53 Ser-15 as indicated. (c, d) Human HEK293T cells were treated with resveratrol, bleomycin, or both as in (a) and probed for gamma -H2AX foci by immunofluorescence. Cell images (291, 267, 216, and 260 cells, respectively) were analyzed for foci using Image J software, and the average number of foci per cell as well as the percentage of cells containing >5 foci were quantitated. (e, f) Human HCT116 cells were incubated with KU-55933, resveratrol, H2O2, and bleomycin as in (a, b) but additional phosphorylation targets were examined using antibodies directed against Smc1, phospho-Smc1 Ser-957, Kap1, phospho-Kap1 Ser-824, Nbs1, phospho-Nbs1 Ser-343, Chk2, and phospho-Chk2 Thr-68 as indicated. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0097969), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human ATM by Western Blot Resveratrol activates ATM in transformed human cell lines.(a, b) Human HEK293T cells were treated with 0.01 mM ATM inhibitor (KU-55933) or vehicle (DMSO) for 1 hour, followed by incubation in media also containing 0.1 mM resveratrol (or DMSO), as indicated. After 30 minutes incubation with resveratrol, H2O2 (0.1 mM) or bleomycin (1 µg/ml) was added as indicated for 30 minutes before harvesting. Western blotting was performed with with antibodies directed against ATM, phospho-ATM Ser-1981, p53, and phospho-p53 Ser-15 as indicated. (c, d) Human HEK293T cells were treated with resveratrol, bleomycin, or both as in (a) and probed for gamma -H2AX foci by immunofluorescence. Cell images (291, 267, 216, and 260 cells, respectively) were analyzed for foci using Image J software, and the average number of foci per cell as well as the percentage of cells containing >5 foci were quantitated. (e, f) Human HCT116 cells were incubated with KU-55933, resveratrol, H2O2, and bleomycin as in (a, b) but additional phosphorylation targets were examined using antibodies directed against Smc1, phospho-Smc1 Ser-957, Kap1, phospho-Kap1 Ser-824, Nbs1, phospho-Nbs1 Ser-343, Chk2, and phospho-Chk2 Thr-68 as indicated. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0097969), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human ATM by Western Blot DEK is necessary for the activation of gamma H2AX after ionizing radiation.(a) MEFs were treated with 10 Gy of gamma ionizing radiation (IR) and stained for gamma H2AX. Representative cross sections from z-stack confocal images are shown. (b) Quantification of cells with >10 gamma H2AX foci in (A). At least 50 cells per time point were counted in each biological replicate, and all foci within the z-stack were quantified. DEK−/− MEFs were unable to activate gamma H2AX above baseline after irradiation. (paired t-test, n = 3, mean ± SEM). (c) Quantification of the average total number of gamma H2AX foci in each cell from (A). (paired t-test, n = 3, mean ± SEM) The number of foci per cell remained unchanged in DEK−/− MEFs after irradiation. (d) Western blot analysis of gamma H2AX in MEFs at 6 hs following irradiation with 10 Gy. (e) Western blot analysis of C33A human cancer cells infected with adenovirus 48 hr prior to receiving 10 Gy IR (see also Supplementary Fig. S1). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28317934), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human Human/Mouse/Rat Phospho-ATM (S1981) Antibody by Western Blot TCL cell lines show intrinsic DNA damage and basal activation of DDR. Healthy T lymphocytes isolated from healthy donors and malignant T cell lines (OCI-Ly12, KARPAS-299, SUP-T1, HH, HD-MAR-2, and JURKAT) were subjected to immunofluorescence for the evaluation of gamma H2AX or 53BP1 foci (A,B). The percentage (%) of cells displaying foci and the number of foci/cell in the subsets of cells with detectable foci are reported in (A,B), respectively. Data are expressed as the mean ± SD of independent experiments. Asterisks indicate statistically significant differences between each cell line and healthy T lymphocytes (* p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant). Representative immunofluorescence images (C,D) of experiments described in (A,B), original microscope magnification 100×. SUP-T1 cells were exposed to increasing concentrations of CHOEP (IC50, 4× and 8×, as in [5]) or to 20 μM etoposide for 3 h. After harvesting, untreated and treated cell lysates were analyzed by Western blot using the indicated antibodies (E). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/35409194), licensed under a CC-BY license. Not internally tested by R&D Systems.