98 | Chapter 4 Figure 5. ATM interacts with FOXO1 but does not phosphorylate FOXO1 on SQ/TQ residues. (A) Proximity ligation assay (PLA) shows the DNA damage-induced interaction between Ser1981phosphorylated ATM and FOXO1 in STI571-treated BV173 BCR-ABL-positive B-ALL cells. Cell were treated as indicated. (B) Constitutive interactions between total ATM (phosphorylated and unphosphorylated) and FOXO1 in STI571-treated BV173 cells. (C) DNA damage-induced interactions between total ATM and Ser15-phosphorylated p53. Red fluorescent foci represent in situ protein proximity (<30 nm). The number of PLA signals per cell in at least 20 cells per condition was quantified. Horizontal lines represent means; statistical significances were determined by oneway ANOVA (*** p<0.001). (D) Western blot analysis of anti-phospho SQ/TQ (ATM/ATR substrate) immunoprecipitated proteins in STI571-treated BV173 cells that were treated as indicated. Input represents total lysates from treated cells. Immunoprecipitation using polyclonal rabbit IgG served as a negative control for each condition. FOXO1 was not detected in the immunoprecitates whereas NBS1 was readily detected in NCS-treated cells. Phosphorylation of SQ/TQ residues on NBS1 was reduced by pretreatment of the cells with KU55933 prior to induction of DNA damage, indicating ATM-dependent phosphorylation. A representative example of two independent immunoprecipitation experiments is shown.
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