Figure 3. Inhibition of DNAPK decreases activation of the S465A mutant.

A) In cells expressing Abl-WT-EGFP, phosphorylation of the biosensor peptide after IR (lane 2) was not significantly diminished using either inhibitor alone (lanes 3 and 4). B) However, in cells expressing Abl-S465A-EGFP, the DNAPK inhibitor NU7206 was sufficient to decrease biosensor phosphorylation levels (lane 4) to pre-IR background control levels (lane 1). B) and D) Over several replicates, cells expressing the Abl-S465A-EGFP and treated with the ATM inhibitor KU55933 also showed some decrease in Abl activation (lane 3) relative to cells with no inhibitor (lane 2)—however this decrease was not statistically significant. Differential ATM and DNAPK inhibition is shown using antiphospho-antibodies to their respective autophosphorylation/activation sites (S1981 for ATM and S2056 for DNAPK). C) and D) Data over three independent experiments were processed as described in Figure 2, averaged, tested for statistical significance relative to non-IR treated controls and plotted (p<0.05 indicated via *). Y-axes represent increase in 4G10/streptavidin signal in samples treated with IR vs. non-IR controls, and are labelled as +IR/-IR. Error bars represent SEM.