Figure 5

ALDOA interacts with DNA-PK- and ATM-containing complexes. (A) 293 T cells were exposed to 6 Gy IR and lysates were prepared for immunoprecipitation with ALDOA antibodies at the indicated timepoints. Eluted proteins and whole cell lysates were separated by electrophoresis and immunoblotted with antibodies against ALDOA and DNA-PK. (B) The reciprocal immunoprecipitation with an anti-DNA-PK antibody was also able to detect an interaction with ALDOA. (C) 293 T cells were transfected with plasmids encoding Flag-ALDOA, 24 h prior to cell lysis and immunoprecipitation with ALDOA antibodies. Associating proteins were immunoprecipitated from 293 T whole cell lysates prepared from cells that had been either left untreated or exposed to 6 Gy IR and harvested after the indicated IR time point. Eluted proteins and whole-cell lysates were separated by electrophoresis and immunoblotted with antibodies against ALDOA and ATM. (D) The reciprocal immunoprecipitation with anti-ATM antibodies was also able to detect an interaction with ALDOA. (E) ALDOA and DNA-PKcs partially colocalise following ionising radiation. U2OS cells were treated or mock-treated with 6 Gy IR and treated with extraction buffer (+ RNase) prior to fixation in PFA at the indicated timepoints. Cells were then stained with the indicated antibodies. (F) ALDOA and P-ATM (S1981) partially colocalise following ionising radiation. U2OS cells were treated or mock-treated with 6 Gy IR and treated with extraction buffer prior to fixation in PFA at the indicated timepoints. Cells were then stained with the indicated antibodies. Unless otherwise stated, data shown are representative of three independent experiments. Immunofluorescence scale bars represent 10 μm.