FIG. 1.
DNA-PKcs interacts with the catalytic and regulatory subunits of PP6. (A) DNA-PKcs was immunoprecipitated from HEK293 cells, and immunoblots were probed with antibodies to DNA-PKcs, PP2Ac, PP4c, or PP6c as indicated. Lanes 1 and 2 contained the supernatants (s) from extracts immunoprecipitated for DNA-PKcs or preimmune serum, respectively. Lanes 3 and 4 contained immunoprecipitates (IP) for DNA-PKcs or preimmune serum as indicated. (B) PP6c was immunoprecipitated from HeLa cells, and samples were analyzed by SDS-PAGE with Coomassie blue staining. PP6c-interacting proteins were identified by mass spectrometry. Indicated are bands identified as DNA-PKcs, PP6c, PP6R1, PP6R2, and PP6R3. Lane 1, preimmune serum; lane 2, PP6c serum. Positions of molecular weight markers (in thousands) are indicated on the left. (C) DNA-PKcs was immunoprecipitated from HEK293 cells as in the experiment for which results are shown in panel A. Immunoblots were probed with antibodies to DNA-PKcs, PP6c, PP6R1, PP6R2, and PP6R3, as indicated. Lanes 1 and 2, supernatants; lane 3, immunoprecipitates with antisera to DNA-PKcs; lane 4, immunoprecipitates with preimmune serum. The asterisk indicates a nonspecific, cross-reacting band in lanes 3 and 4. (D) PP6c, PP6R1, PP6R2, and PP6R3 were immunoprecipitated from HEK293 cells as in the experiments for which results are shown in panels A and C, and immunoblots were probed for DNA-PKcs. (E) PP6c, PP6R1, PP6R2, and PP6R3 were expressed in bacteria with N-terminal GST tags, immobilized on glutathione-Sepharose beads, and incubated with 5 μg purified DNA-PKcs protein (in the absence of Ku). Beads were washed as described in Materials and Methods, and proteins were analyzed by SDS-PAGE, followed by immunoblotting with an antibody to DNA-PKcs (upper panel). Lane 1, DNA-PKcs with beads alone; lane 2, DNA-PKcs plus GST-bound beads; lanes 3 to 6, DNA-PKcs plus GST-PP6c, PP6R1, PP6R2, or PP6R3 as indicated. The SDS-PAGE gel was stained with Coomassie blue to show protein loading (lower panel). The positions of migration of PP6 subunits are shown on the right. The asterisk in lane 3 represents a contaminating band.