FIG. 3.
The catalytic and regulatory subunits of PP6 subunit localize to the nucleus, and their association with DNA-PKcs does not change after IR. (A) HEK293 cells were either left untreated (−) or treated with 10 Gy IR (+) and were left to recover for the times indicated. Cells were fractionated into cytoplasmic (S10) (lanes 1 to 3) or nuclear (P10) (lanes 4 to 6) extracts as described in Materials and Methods. Fifty micrograms of protein was run on SDS-PAGE gels and probed for PP6c, PP6R1, PP6R2, PP6R3, DNA-PKcs, GAPDH, or Ku80 as indicated. (B) P10 extracts were prepared and analyzed as for panel A. Levels of PP6c (gray bars) and PP6R1 (black bars) were normalized to nuclear Ku80 protein levels (set at 1). unIR, unirradiated. (C) DNA-PKcs was immunoprecipitated from detergent lysates from either unirradiated cells (lane 1) or cells that had been irradiated with 10 Gy and harvested 2, 4, or 8 h later as indicated (lanes 2 to 4). Immunoprecipitates were run on SDS-PAGE gels and were immunoblotted for DNA-PKcs, PP6c, PP6R1, PP6R2, or PP6R3 as indicated. Lane 5, preimmune sera (control); lane 6, input (representing 25 μg of total protein from the whole-cell extract). Note that longer exposures showed the presence of DNA-PKcs in the input lane (lane 6).