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. 2009 Dec 17;38(6):1821–1831. doi: 10.1093/nar/gkp1164

Figure 5.

Figure 5.

Exo1 interacts with ATM and phosphorylation of Exo1 on S714 is dependent upon ATM. (A) HeLa cells were pretreated or mock-treated with the ATM inhibitor, KU55933 (10 µM) 1 h prior to treatment with 6 Gy IR and fixed at the indicated time points. Cells were immunostained with the indicated antibodies. The scale bar represents 10 µm. (B) Top panels, HeLa cells were transfected with Flag-Exo1 and treated with 40 J/M2 UVC, 6 Gy IR or 2 mM HU and extracts taken after the indicated time. Immunoprecipitations were carried out using Flag M2 Beads. Immunoprecipitates were immunoblotted with the indicated antibodies. Bottom panels, HeLa cells were treated as above and immunoprecipitated using ATM antibodies. Immunoprecipitates were immunoblotted with the indicated antibodies. (C) Exo1 interacts with GST-ATM fragment 8 (amino acids 1764–2138). Recombinant GST-ATM fragments representing the full length of ATM were used in pull-down assays. Total cell extracts from HeLa cells expressing Flag-Exo1 were mixed with glutathione agarose beads containing GST-ATM fusion proteins. Bound proteins were analysed by immunoblotting with Flag antibodies (top panel) and levels of GST-ATM fragments were detected by coomassie staining (bottom panel).