ATM autophosphorylation requires both E2F1 and MRE11A in BIN1-deficient cells.
A, Western blotting (left) and densitometry analysis (right) of the amount of pan-ATM and pSer-1981 ATM proteins in DU145±sh-BIN1 cell lines cultured under optimal conditions. β-Actin was used as the loading control. B, Western blot analysis of pan-ATM, pSer-1981 ATM, E2F1, and BIN1 in DU145±sh-BIN1 cell lines transiently transfected with si-E2F1 or si-Cont. GAPDH was used as the loading control. C, Western blot analysis of pan-ATM, pSer-1981 ATM, MRE11A, and BIN1 in the human fibroblast cell line (GM00637) transiently transfected with si-BIN1, si-MRE11A, or si-Cont. β-Actin was used as the loading control. D, in human normal (GM00637) fibroblasts cultured under optimal conditions, the effects of transient transfection with si-BIN1 alone, si-MRE11 alone, and the combination of these two siRNAs on pSer-1981 ATM were analyzed with in situ immunofluorescence microscopy, probed with an anti-phospho-ATM (Ser-1981)-specific antibody (red). Nuclei were counter-stained with DAPI (blue). Scale bar, 10 μm.