ATM is the gene that is mutated in ataxia telangiectasia, an autosomal recessive hereditary disease that is characterized by delayed motor and sexual development, weakened immunity, multiple skin changes, increased sensitivity to ionizing radiation, and increased risk of developing certain cancers. The protein encoded by ATM is a phophoinositide-3-kinase-related protein kinase that is found in the nucleus and is responsible for early responses to the changes in chromatin structure caused by DNA double-strand breaks. Inactive wild type ATM protein forms homodimers or higher order multimers in which the C-terminal kinase domains are inhibited. It has been proposed that multimeric ATM is recruited to DNA double-strand breaks through interactions between its N-terminal HEAT domains and specific chromatin protein complexes. The inhibition of the kinase domains is lifted, resulting in auto-phosphorylation of the ATM at a single serine site and disruption of the multimers. The phosphorylated state of ATM is a rapid and highly sensitive indicator that cells have been exposed to agents that cause DNA double-strand breaks. The kinase domains of the resulting ATM monomers are accessible to a wide variety of substrates that are involved in DNA repair, cell-cycle regulation, and apoptosis. Thus ATM is a central regulator of cellular responses to ionizing radiation, and cells that lack ATM undergo radioresistant DNA synthesis and are resistant to γ radiation-induced apoptosis. The K88-534 monoclonal antibody recognizes human ATM phosphorylated at serine 1981 (S1981) and mouse ATM phosphorylated at its orthologous site, S1987. Immunofluorescent detection of ATM phosphorylation in human fibrosarcoma cells. HT-1080 cells (ATCC CCL-121) were seeded in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) at ~ 10,000 cells per well. After overnight culture, DNA damage was induced by exposing the cells to bleomycin sulfate (250 units/mL) in serum-free medium for 2 hours at 37 °C (left panel) or the cells were untreated (right panel). To allow time for DNA repair processes to occur, the cells were washed, replenished with fresh complete medium, and returned to the incubator for 30 minutes. After treatment, cells were stained using the alcohol perm protocol and the Purified Mouse anti-ATM (pS1981) (pseudo-colored green), and counter-stained with Hoechst 33342 (pseudo-colored blue) according to the Recommended Assay Procedure. The second-step reagent was Alexa Fluor® 488 goat anti-mouse Ig (Invitrogen). Confocal images were captured on a BD Pathway™ 855 Bioimaging System using a 20x (0.75 NA) objective. Five sections, separated by 1 μm each, were captured and collapsed for viewing purposes using BD Attovision™ software. 560007 Rev. 1 Page 1 of 2