Figure 3

SMAR1 interacts with γH2AX and is phosphorylated by ATM at serine 370 upon IR. (a) Immunofluorescence analysis of HCT116 cells for SMAR1 (red) and γH2AX (green) upon IR (10 Gy) at indicated time points. DAPI (blue) was used to stain nucleus. (b) IP assay to check interaction between γH2AX and SMAR1 upon IR (10 Gy, 2 h). Irradiated HCT116 cell lysates were immunoprecipitated with SMAR1 and the eluates were immunoblotted with the indicated antibodies. (c) Immunofluorescence analysis of SMAR1 (red) and γH2AX (green) in HCT116 cells that were either left untreated or pretreated with KU-55933 (10 μM, 2 h) before IR (10 Gy). (d) A Schematic representation of SMAR1 protein sequence showing different regions along with a potential ATM phosphorylation site serine 370 in wild-type SMAR1(WT), which is substituted by alanine in mutant form of SMAR1 (Mut). (e) ATM-mediated phosphorylation of SMAR1 was studied by performing in vitro kinase assay. Immunoprecipitated ATM from irradiated cell lysate was incubated with 1 μg each of recombinant GST-SMAR1 (1–548), various GST-tagged truncations of SMAR1 and GST alone. Phosphorylation of SMAR1 was observed by autoradiography. The panel below depicts coomassie staining of all recombinant proteins used for kinase assays. (f) Immunoblot analysis of SMAR1 phosphorylation in HCT116 cells upon irradiation (10 Gy) using anti-phospho-SMAR1 antibody at indicated time points. (g) IP assay to study the association of SMAR1 with ATM in irradiated HCT116 cells (10 Gy, 2 h) transfected with either Flag-SMAR1 (F-SM) or Flag-mutant (F-Mut). Whole-cell lysates were immunoprecipitated with anti-Flag antibody followed by immunoblotting with indicated antibodies. (h and i) Immunoblotting of phospho-SMAR1 and indicated proteins in MEFs from ATM^+/+ and ATM^−/− mice (h), and A-T cell lines such as C3ABR and L3 (i) upon IR (10 Gy, 2 h). (j) Western blot analysis of SMAR1 and mentioned proteins in chromatin fractions from HCT116 cells that were treated with KU-55933 (10 μM, 2 h) before irradiation