Fig. 7

CBP binds to and acetylates ATM in response to DNA damage. a Immunoprecipitation (IP) of CBP from T47D cells treated with Doxorubicin (DOX) for 8 h. The cell extracts were pretreated with and without ethidium bromide (EtBr). Immunocomplexes were analyzed by immunoblotting using the indicated antibodies. b Band quantification of CBP, ATM, and p-ATM levels in both immunoprecipitated and input samples. c Left panel: Proximity ligation assay (PLA) for CBP and ATM colocalization (red) in T47D cells after ionizing radiation (IR) or after incubation with 5 µM Cisplatin (CIS). DAPI staining (blue) shows the nucleus. Scale bar, 100 µm. Right panel: Bar graphs representing the quantification of PLA signals from 100 cells. d T47D cells transfected with the indicated siRNAs for 48 h were incubated with DOX. Cell lysates were subjected to immunoprecipitation using ATM antibody. The acetylation level was determined by immunoblotting using anti-acetyl lysine (anti-acK) antibody. e Band intensities for acetyl lysine in ATM-immunoprecipitated samples, and for ATM and CBP in input samples, normalized to β-actin. f T47D cells were incubated with CBP HAT or Tip60 HAT inhibitors, followed by DOX treatment. After 8 h, the cells were collected, and immunoprecipitation was performed using anti-ATM antibody followed by immunoblotting with anti-acetyl lysine (anti-acK) antibody. g Densitometric quantification of acetyl-ATM levels in T47D cells treated with the indicated inhibitors and DOX. h CBP was immunopurified from T47D cells treated with 8 µM of CBP HAT inhibitor or 25 µM of Tip60 HAT inhibitor, followed by DOX treatment. ATM acetylation activity was assessed by measuring the release of free CoA via absorbance at 570 nm in the presence of tetrazolium dye. Data are represented as mean ± SEM, n = 3. P < 0.05 is considered significant, Mann–Whitney U test