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. 2012 Aug 24;4(5):304–315. doi: 10.1093/jmcb/mjs048

Figure 3.

Figure 3

ATM phosphorylates Snail on Serine 100. (A) The ATM phosphorylation consensus sequence of Snail in human, rat, mouse, and Xenopus. (B) Snail was immunoprecipitated from mock- or CPT-treated MDA-MB-231 cells, followed by immunoblotting with indicated antibodies. (C) Flag-tagged WT or the serine 100 to alanine mutant form (S100A) of Snail was stably transfected in HEK293 cells. Cells were treated with CPT, and lysates were immunoprecipitated by anti-Flag beads followed by immunoblotting with indicated antibodies. (D) Isogenic HeLa cells with proficient or deficient ATM were treated with mock or CPT followed by immunoprecipitating Snail and immunoblotting pS100-Snail. (E) MDA-MB-231 cells and HEK293 cells were treated with 6 Gy IR, incubated for 2 h, immunoprecipitated for Snail, and immunoblotted to detect pS100 Snail expression levels. (F) The in vitro kinase assay was performed using ATM fragments (either GST-tagged N-terminal a.a. 248-522 or C-terminal fragments of ATM a.a. 2709-2964) in the presence of non-phosphorylated Snail peptides. The blot was stained by Coomassie blue and immunoblotted with the anti-phospho-S100 Snail antibody.