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. 2011 Aug 8;39(21):9224–9237. doi: 10.1093/nar/gkr647

Figure 4.

Figure 4.

IR-induced phosphorylation on serine 114 is ATM dependent, while phosphorylation at serine 126 is both ATM and DNA-PK dependent. (A) Cells were either untransfected (u, lane 1), or transfected with vector (v, lane 2), PNKP-S114A-V5 (lane 3) or wt-PNKP-V5 (lanes 4–8). Twenty-four hours after transfection, cells were pretreated with 10 µM NU7441 (DNA-PK inhibitor) (lanes 1, 2, 3 and 7), DMSO control (lane 4), 2 µM NU7441 (lane 5) or 5 µM NU7441 (lane 6) for 2 h prior to irradiation (2 Gy, lanes 1–7). The sample in lane 8 was not irradiated. Thirty minutes after irradiation, PNKP was immunoprecipitated and analyzed by western blot using the phospho-specific antibody to S114 or V5 for total PNKP as above. (B) Cells were pre-treated with the indicated concentrations of the ATM inhibitor KU55933 for 2 h, then irradiated (2 Gy) and analyzed exactly as described above. PNKP was immunoprecipitated and observed by western blotting with either the phospho-specific antibody to S114 or V5 for total PNKP. As above, u and v represent untransfected (lane 1) and vector (lane 2). S114A refers to the S114A mutant of PNKP (lane 3). (C) Cells were pre-treated with either 10 µM NU7441 or 5 µM KU55933 and either unirradiated (lane 3) or irradiated at 20 Gy (lanes 1, 2, 4–8) and harvested after 1 h (lane 4) or 4 h (lanes 1, 2, 5–8). PNKP was immunoprecipitated and analyzed by western blotting with either the phosphospecific antibody to S114 or V5 for total PNKP as above. (D) Cells were pre-treated with NU7441 and/or KU55933 as in (C), then irradiated (10 Gy). PNKP was immunoprecipitated after 30 min and analyzed by western blot as described, but using a phospho-specific antibody to serine 126.