Fig. 1

Co-immunoprecipitation assays for the interaction of ATM and p400. a HEK293T cells were transfected with pcDNA-Flag-ATM, CβS-HA-TIP60, CβF-TIP60 and CβS-HA-p400 in various combinations. Both input and anti-HA immunoprecipitation samples were analysed on an immunoblot using anti-HA and anti-Flag antibodies while the input samples were additionally examined with anti-actin antibody as a loading control. Immunoglobulin heavy chain is shown in the right panel of immunoprecipitation analysis. * non-specific band. b HEK293T cells were transfected with plasmid expressing Flag-p400 (lane 2). The whole cell extracts were subjected to immunoprecipitation with M2 agarose and the immunoblot was examined with anti-ATM and anti-Flag antibodies. The 5% (v/v) of input whole cell extract was also analysed by immunoblotting in the bottom panels to show the total ATM level and β-actin. c HEK293T cells were transfected with pcDNA-Flag-ATM, CβS-HA-TIP60, CβS-HA-GAS41, CβS-HA-BAF53 in various combinations. Both input and immunoprecipitation samples were examined by immunoblotting. d Co-immunoprecipitation between ATM and p400 in the presence and absence of DNA damage. HEK293T cells were transfected in duplicate with pcDNA-Flag-ATM alone or together with CβS-HA-TIP60 or CβS-HA-p400. Transfected cells were incubated in the presence and absence of bleomycin for 4 h before being analysed by immunoblotting. To assess the DNA damage by bleomycin, histones were separately prepared by acid extraction and the immunoblot was analysed by anti-γ-H2A.X (phospho-S139) antibody