In vitro DNA-PKcs kinase assay

Each kinase reaction contained five times kinase buffer (125 mM Tris–HCl, pH 7.9, 125 mM MgCl₂, 5 mM DTT, 125 mM KCl, and 50% glycerol), 100 ng of sonicated herring DNA, 20 nM Ku70/80, 0.16 μM [γ-³²P] ATP (6000 Ci/mmol), 1 μg biotin-labeled H2AX (biotin-AVGKKASQASQEY) and 10 μg of nuclear extract from HCT116 DNA-PK_cs +/+, +/−, −/−, or KD/− cell lines. The final volume was brought to 10 μl. Reactions were incubated for 30 min at 30°C and terminated by the addition of 1 μl 0.5 M ethylenediaminetetraacetic acid (EDTA). The biotinylated-H2AX peptide was captured using a SAM2 Biotin Capture Membrane (Promega) and the membrane was washed following the manufacturer's suggested protocol. H2AX phosphorylation was detected by PhosphoImager analysis (Amersham Biosciences) and scintillation counting. Background phosphorylation of H2AX was observed in the −/− cell line and this was subtracted from the other samples’ readouts. 100% kinase activity was normalized using the +/+ cell lysate results. The reported results are derived from three independent experiments.