FIG. 2.

Transfectants expressing multiple phosphorylation site substitutions express normal levels of DNA-PK activity. (A) Whole-cell extracts (500 μg) prepared from V3 cells transfected with vector alone, wild-type DNA-PKcs, mutant ABCD, or mutant ABCDE were assayed for DNA-PK activity as described in Materials and Methods. Phosphorylation of the p53 substrate was assessed after 15, 30, and 120 min as indicated. Each cell extract was tested in duplicate, and three independent extracts were tested for each cell line. Error bars, standard deviations. (B) Whole-cell extracts (250 μg) from V3 cells transfected with vector alone, wild-type DNA-PKcs, or mutant ABCDE were assayed for the capacity to phosphorylate no substrate (lanes 1 to 3), recombinant XRCC4 (lanes 4 to 6), or recombinant Artemis (lanes 7 to 9). (C) DNA-cellulose fractions from 500 μg of whole-cell extracts of V3 cells expressing wild-type DNA-PKcs (lane 1), vector alone (lane 2), or mutant ABCDE (lane 3) were incubated in protein kinase buffer with [γ-³²P]ATP. Subsequently, bound proteins were analyzed by SDS-4% PAGE and autoradiography.